v-mos Oncoproteins Affect the
Nuclear Retention and Reutilization
of Glucocorticoid Receptors
Ming Qi, Beverly J. Hamilton, and Donald DeFranco
Department of Biological Sciences
University of Pittsburgh
Pittsburgh, Pennsylvania 15260
Expression of the p85gag"mos oncoprotein in temperature sensitive transformed 6m2 cells results in desensitization of glucocorticoid induction of metallothionein-1 mRNA. Indirect immunofluorescence
analyses demonstrate that hormone insensitivity in
v-mos transformed cells is associated with inefficient nuclear retention of glucocorticoid receptor
(GR) protein. Desensitized receptors that accumulate in the cytoplasm of transformed 6m2 cells do
not regain the capacity for hormone-dependent nuclear translocation after turnover of the thermo-labile p85gagmos oncoprotein. Although ligand induced
down-regulation of immunoreactive GR protein occurs in transformed 6m2 cells, desensitized receptors appear to retain some capacity to bind hormone
in vivo. Thus alterations in the intracellular partitioning of GR protein in v-mos-transformed cells result
in the generation of a novel desensitized receptor
that is apparently trapped in the cytoplasm and
incapable of being reutilized. (Molecular Endocrinology 3: 1279-1288, 1989)
different cell-types (13-16). The binding of ligand to a
steroid receptor protein can therefore be envisaged as
triggering a chain of events in the steroid hormone
signal transduction pathway that ultimately leads to
modulation of specific gene activity (1). However, it is
not clear that receptor function and activity within a cell
effectively terminates after its interaction with the transcriptional machinery. Thus, although the equilibrium
subcellular distribution of GR protein is shifted to the
nuclear compartment after ligand binding and activation
(12), receptors may be exported from the nucleus in
the continued presence of hormone and subsequently
reutilized via recycling pathways (17-19). Such putative
GR recycling pathways implicate the existence of novel
intermediate receptor forms, both nuclear and cytoplasmic, for which no direct physical evidence has been
provided (20). Despite uncertainty concerning the redistribution and reutilization of nuclear GR protein via
recycling pathways, it has been demonstrated that GR
protein exit the nuclear compartment after hormone
withdrawal (11, 21). Although recycling and hormone
withdrawal both involve the redistribution of nuclear
receptors, it has not been established whether these
processes are mediated via the same or independent
mechanisms. Nuclear export of GR protein may be
energy dependent, since depletion of cellular ATP levels
in cultured rat thymocytes leads to efficient nuclear
retention of an unliganded null receptor (22).
Complex physiological responses to steroid hormones are most likely the result of combinatorial interactions between multiple gene regulatory factors (1,
23), and not strictly governed by the presence of functional receptor systems (24, 25). In addition to normal
physiological influences on steroid hormone responsiveness, effects can also be exerted by abnormal
changes in cellular physiology. The influence of oncogenic transformation on steroid hormone responsiveness was first noted by the inhibition of glucocorticoidinduced differentiation of bone marrow preadipocytes
upon infection with Moloney murine sarcoma virus (26).
Subsequently, p37mos, a 37-kilodalton (kDa) cytoplasmic oncoprotein encoded by Moloney murine sarcoma that possesses an associated protein-serine/threonine kinase activity (27, 28), was shown to inhibit
glucocorticoid induction of mouse mammary tumor vi-
INTRODUCTION
Steroid hormone receptors are a class of enhancerbinding proteins that modulate transcription through
sequence-specific interactions with hormone response
elements (1, 2). As members of a supergene family of
ligand-activated gene regulatory proteins (3, 4), steroid
receptors are structurally related, being comprised of
distinct, independently functional domains that specify
ligand binding, DNA binding, and transcriptional activation activities (5-9).
The interactions of glucocorticoid receptor (GR) protein with various intracellular components are altered
after ligand binding as evidenced in part, by the dissociation of receptors from oligomeric complexes (10) and
the acquisition of the capacity for tight nuclear binding
(11, 12). In addition, agonist-bound receptors once
activated, are subject to more rapid turnover in many
0888-8809/89/1279-1288S02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
1279
MOL ENDO-1989
1280
rus transcription (29). We have recently uncovered effects of the p85gagmos oncoprotein on glucocorticoid
induction of metallothionein-1 (Mt-1) gene expression
using a rat cell line (6m2 cells; 30) that is temperature
sensitive for p85gag"mos expression, and thereby transformation (31). In transformed 6m2 cells, glucocorticoid
induction of Mt-1 mRNA is only transient, and not
maintained to the same extent as in nontransformed
6m2 cells (32).
In this report we have set out to determine whether
the inability of a glucocorticoid response to be maintained in p85gagmos transformed cells represents a desensitized response, and whether oncoprotein effects
are exerted on GR activity. We report here that p85gag"
mos
affects both the nuclear retention and reutilization
of GR protein. Our results imply that desensitization of
GR protein by p859agmos reflects either abnormal nuclear
export of receptors or abnormal processing of recycled
cytoplasmic receptors.
RESULTS
Dexamethasone Induction of Mt-1 mRNA is
Desensitized in p85gag"""-Transformed 6m2 Cells
In NRK cells transformed by p85gagmos (6m2 cells; 30),
dexamethasone induction of Mt-1 mRNA is only transient, and within 24 h of hormone treatment, Mt-1
mRNA levels return to that observed in unstimulated
cells (32). In order to determine whether this transient
induction represents a desensitized response, the effectiveness of a secondary hormone treatment in inducing Mt-1 mRNA levels was tested in transformed 6m2
cells that were pretreated with dexamethasone for 24
h. Total RNA was isolated at various times during this
secondary hormone treatment and Mt-1 mRNA levels
analyzed by Northern blot analysis. As shown in Fig. 1,
Mt-1 mRNA levels were not significantly increased at
any time during a secondary dexamethasone treatment
of transformed 6m2 cells. These results demonstrate
that metabolism of dexamethasone does not account
for transient induction of Mt-1 mRNA, which was expected given the in vivo stability of this synthetic hormone. In contrast to the ineffectiveness of a secondary
dexamethasone treatment, CdCI2 was still able to induce Mt-1 mRNA in transformed 6m2 cells that had
been pretreated with dexamethasone for 24 h (Fig. 1).
A decrease in induced Mt-1 mRNA levels observed
after a prolonged (24 h) CdCI2 treatment had been
observed previously in both transformed and nontransformed 6m2 cells (32), and is not indicative of v-mos
effects on heavy-metal induction of Mt-1 mRNA. Therefore, desensitization of glucocorticoid induction does
not affect induction of Mt-1 mRNA by an agent that
acts through an independent signal transduction pathway (33). Likewise, since dexamethasone was continuously present throughout the period of induction by
CdCI2 (Fig. 1), accelerated decay of Mt-1 mRNA in
hormone-treated transformed 6m2 cannot completely
Vol 3 No. 8
account for transient dexamethasone induction of Mt1 mRNA.
GR Protein is not Efficiently Retained in the
Nucleus of Dexamethasone-Treated p85gagmosTransformed 6m2 Cells
During a period of desensitization, some component of
the hormone induction system becomes refractory or
insensitive to a continued hormonal stimulus. Since
nonfunctional GR protein can be associated with both
nuclear and cytoplasmic compartments (34, 35), the
assessment of v-mos effects on desensitization of glucocorticoid responsiveness requires a consideration of
the subcellular localization of receptors. Indirect immunofluorescence (IIF) was therefore used to visualize the
subcellular distribution of GR protein in nontransformed
and v-mos-transformed cells. Picard and Yamamoto
(12) have shown using IIF that in CV-1 and COS7
monkey kidney cells, unliganded GR protein localized
predominately to the cytoplasmic compartment under
certain culture conditions. In our experiments using
similar cell culture and fixation conditions (see Materials
and Methods), and the BuGR2 monoclonal antibody
raised against rat GR protein (36), we have also observed a predominant cytoplasmic localization of unliganded receptors (Fig. 2, and all subsequent IIF figures). Thus, our IIF experiments examine v-mos effects
on hormone-dependent nuclear transfocation of GR
protein (12).
For our first set of IIF experiments, the subcellular
distribution of GR protein in 6m2 cells was analyzed
during a time course of dexamethasone treatment of
temperatures permissive (31 C) and nonpermissive
(39C) for p85ga9mos-transformation (31). As shown in
Fig. 2, a 30-min dexamethasone treatment of nontransformed (39 C) 6m2 cells led to the predominant nuclear
localization of GR protein. Importantly, receptors were
retained in the nucleus of nontransformed 6m2 cells
after 12-24 h of hormone treatment (Fig. 2, Table 1).
However, in dexamethasone-treated transformed
(31 C) 6m2 cells, the subcellular distribution of glucocorticoid receptors during a dexamethasone time
course was quite different. As shown in Fig. 2, although
receptors efficiently translocated to the nucleus within
30 min of hormone treatment, they were not retained
there. In some cells, receptors were apparently excluded from the nucleus after only 4 h of hormone
treatment (Fig. 2, Table 1). Strikingly, after 12-24 h of
dexamethasone treatment, receptors were completely
excluded from the nucleus and were exclusively cytoplasmic in transformed 6m2 cells (Fig. 2, Table 1). This
cytoplasmic staining pattern was particularly uniform as
no nuclear staining above background could be detected in any transformed 6m2 cells examined after this
length of hormone treatment. In addition, receptors
remained cytoplasmic in transformed 6m2 cells after
prolonged (>24 h) hormone treatment despite the frequent addition of fresh dexamethasone containing medium (not shown). These results lend further support to
1281
v-mos Effects on GR
Actin
MT-1
0
4
1 2 2 4 2 8 2 8 3 6 3 6 3 6 4 8 4 8 4 8
0
4
4
-
+
+
-
-
+
1 2 1 2 1 2 2 4 2 4 2 4
+
.
-
+
+
+
-
-
+
+
Hrs. in 1° dex
Hrs.
2° dex
CdCl2
Fig. 1. Dexamethasone Induction of Mt-1 mRNA Levels is Desensitized in p859a9~mos-Transformed 6m2 Cells
6m2 cells grown at 31 C were treated with 10~6 M dexamethasone (dex) for the lengths of time indicated (1° dex). After 24 h of
dex treatment, some cells were given either an additional dose of dex (2° dex), or 10"6 M CdCI2 and incubated for the lengths of
time indicated. Total RNA (5 ng) was electrophoresed, blotted, and hybridized to 32P-labeled chicken /8-actin (top) or mouse Mt-1
(bottom) DNA probes. Autoradiographs were exposed for 8 h (top) or 5 days (bottom).
the conclusion that oncoprotein effects are not due to
altered metabolism of dexamethasone.
It must be emphasized that the p859agmos oncoprotein
is present in transformed 6m2 cells (31 C) before hormone treatment. Thus, oncoprotein effects are not exerted upon hormone dependent nuclear translocation
of previously unstimulated cytoplasmic GR protein.
Rather, it is only after hormone treatment 6m2 cells
(>4 h) that cytoplasmic receptors accumulate that are
incapable of translocating to the nucleus even in the
continuous presence of hormone. Since the accumulation of GR protein in the cytoplasm of hormone treated
transformed 6m2 cells correlates with desensitization
of a glucocorticoid response (Fig. 1), and to simplify
further discussions, we will refer to this putative novel
form of receptor as desensitized.
Inefficient Nuclear Retention of GR Protein in v-mosTransformed NRK Cells is not Temperature
Dependent
Transient glucocorticoid induction of Mt-1 mRNA in vmos-transformed NRK cells is not temperature dependent (32), and observed in a revertant cell line derived
from 6m2 cells that is transformed at both 31 C and
39 C (54-5A4 cells; 37). In 54-5A4 cells, a 100-kDa gagmos fusion oncoprotein, p1009a9"mos, is expressed irrespective of growth temperature (37). Therefore, we
have used transformed 54-5A4 cells, and nontransformed NRK cells to corroborate v-mos effects on the
inefficient nuclear retention of GR protein. Figure 3
displays the results of IIF analysis of GR protein during
a time course of dexamethasone treatment in transformed 54-5A4 cells grown at 39 C and nontransformed
NRK cells grown at 31 C. Consistent with our previous
findings, receptors were not efficiently retained in the
nucleus of hormone treated p1009agmos-transformed
cells (Fig. 3, Table 1). Thus, although efficiently translocated to the nucleus within 30 min of dexamethasone
treatment, GR protein was excluded from the nucleus
and exclusively cytoplasmic in 54-5A4 cells after 12 h
of hormone treatment (Fig. 3, Table 1). The initial nuclear translocation of receptors in v-mos-transformed
NRK cells may be more efficient at 39 C than 31 C, as
nuclear staining of receptors 30 min after hormone
treatment was more distinct in 54-5A4 cells grown at
39 C (Fig. 3) than in 6m2 cells grown at 31 C (Fig. 2).
In nontransformed NRK cells, glucocorticoid receptors
efficiently translocated to the nucleus within 30 min of
dexamethasone treatment, and as observed in nontransformed 6m2 cells (39 C, Fig. 2), were retained
there for as long as 12-24 h in the presence of hormone
(Fig. 3, Table 1). The same pattern of inefficient nuclear
retention of receptors specifically in hormone treated vmos-transformed NRK cells was confirmed by IIF experiments performed with NRK and 54-5A4 cells grown
at 39 C and 31 C, respectively (Table 1). Thus, inefficient nuclear retention of GR protein after dexamethasone treatment is a property associated with v-mostransformation of NRK cells and is not temperature
dependent.
Redistribution of Nuclear GR Protein in
Dexamethasone Treated p85gagmos-Transformed
6m2 Cells Does not Require Concomitant Protein
Synthesis
Glucocorticoid activation of transcription can occur in
the absence of concomitant protein synthesis (1). Likewise, upon hormone withdrawal, depletion of nuclear
GR protein and replenishment of unactivated cytoplasmic receptors does not require protein synthesis
(18, 21). In fact, much of the evidence for recycling and
reutilization of GR protein is based upon the redistri-
Vol 3 No. 8
MOL ENDO-1989
1282
Dex Time(hrs)
Table 1. Subcellular Localization of GR
Localization
Time in Dex
(h)
Transformed Cells (6m2,
31 Ca; 54-5A4, 31 C, 39
C)
+
+
0
0.5
4
12
24
0.5
N b , N > C to N = C
N > C, N = C some C
C
C
Nontransformed
Cells (6m2, 39
C ; NRK, 31 C,
39 C)
C
N > C to N
N
N
N to N > C
Immunofluorescence analyses were performed as described
in Materials and Methods. C and N, Cytoplasmic and nuclear
fluorescence, respectively; N > C, predominately nuclear fluorescence accompanied by some cytoplasmic staining; N =
C, equal nuclear and cytoplasmic staining.
a
Cells were also incubated in the presence of 5 Mg/ml cycloheximide.
6
Complete nuclear fluorescence in transformed cells after a
30-min incubation with dexamethasone (Dex) was only observed in 54-5A4 cells cultured at 39 C.
4
12
Dex Time(hrs)
31 °C
39°C
Fig. 2. GR Protein is not Efficiently Retained in the Nucleus of
Dexamethasone (dex) Treated p859ag"mos-Transformed 6m2
Cells
Immunofluorescence micrographs of transformed (31 C) and
nontransformed (39 C) 6m2 cells grown in the presence of
10" 6 M dex for the lengths of time indicated.
bution of receptors in the absence of de novo receptor
synthesis (17,18). In order to establish that cytoplasmic
desensitized GR protein in transformed 6m2 cells derive
from the recycling of previously nuclear-bound receptors and not de novo synthesis of receptors, the subcellular distribution of receptors was analyzed in hormone treated cells cultured in the continuous presence
of cycloheximide. As shown in Fig. 4, cycloheximide
treatment which caused a greater than 90% reduction
in total protein synthesis (not shown) did not affect
hormone-dependent nuclear translocation of GR protein in transformed (31 C) and nontransformed (39 C)
6m2 cells. In addition, receptors were efficiently retained in the nucleus of nontransformed 6m2 cells after
12-24 h of hormone treatment in the presence of
cycloheximide (Fig. 4, Table 1). In transformed 6m2
cells that were treated with cycloheximide, GR protein
was not efficiently retained in the nucleus, and redistributed to the cytoplasm within 12 h of hormone treatment
(Fig. 4). Thus, replenishment of cytoplasmic receptors
which followed the depletion of nuclear receptors in
hormone treated transformed 6m2 cells, occurs in the
0.5
12
Fig. 3. Inefficient Nuclear Retention of GR Protein in v-mosTransformed NRK Cells is not Temperature Dependent
Immunofluorescence micrographs of transformed (T) 545A4 (39 C) and nontransformed (NT) NRK (31 C) cells grown
in the presence of 10r6 M dexamethasone (dex) for the lengths
of time indicated.
v-mos Effects on GR
1283
Transformed 6m2 cells (31 C) were treated with dexamethasone for 24 h in order to derive cytoplasmic
desensitized receptors, then shifted to 39 C and maintained in hormone containing medium. At various times
after the temperature upshift, the subcellular distribution of GR protein was visualized by I IF. As shown in
Fig. 5, preexisting cytoplasmic desensitized receptors
in 6m2 cells were not capable of translocating to the
nucleus even after hormone-treated cells had been
shifted to 39 C for 12 h. Thus, it appears that the
presence of p859a9"mos is not required to maintain, for at
least 12 h, the cytoplasmic localization of desensitized
GR protein in hormone-treated 6m2 cells. Desensitized
receptors appear to be trapped in the cytoplasm of
p859ag"mos-transformed cells, and cannot be reutilized
after turnover of this thermo-labile oncoprotein.
Cytoplasmic receptors in unstimulated transformed
6m2 cells can translocate to the nucleus if hormone is
added at the onset of a temperature upshift (Fig. 5).
Thus, shifting 6m2 cells from 31 C to 39 C did not
Dex Time(hrs)
+
0.5
Dex Time(hrs)
12
31 "C
6m2,T
39"C
6m2,NT
Fig. 4. Redistribution of GR Protein in Dexamethasone (dex)
Treated p859a9mos-Transformed 6m2 Cells Does not Require
Concomitant Protein Synthesis
Immunofluorescence micrographs of transformed (T, 31 C)
and nontransformed (NT, 39 C) 6m2 cells grown in the presence of 10~6 M dex and 5 M9/ m| cycloheximide for the lengths
of time indicated.
0.5
presumed absence of de novo receptor synthesis. We
therefore conclude that desensitized GR protein that
accumulates in the cytoplasm of hormone treated vmos-transformed cells derives from the redistribution
or recycling of preexisting nucleus receptors.
Desensitized Cytoplasmic GR Protein in
Temperature Sensitive p85989 """-Transformed 6m2
Cells Cannot be Reutilized after a Shift to the
Nonpermissive Temperature
p85ga9"mos is a thermo-labile oncoprotein and not detected in 6m2 cells 4 h after a shift from permissive to
nonpermissive temperatures (38). The protein kinase
activity associated with p859agmos is also thermo-labile
and rapidly inactivated in vitro with a t>/2 of approximately
3 min after a shift from 33 C to 39 C (39). Given these
properties of p859agmos, temperature upshift experiments provide a useful approach for determining
whether the long term maintenance of desensitized GR
protein in transformed 6m2 cells requires the continuous presence of the oncoprotein.
12
31"C:
t39"C:+Dex
31"C+Dex,24h
f39"C:+Dex
Fig. 5. Desensitized Cytoplasmic GR Protein in TemperatureSensitive p859agmos-Transformed 6m2 Cells cannot be Reutilized after a Shift to the Nonpermissive Temperature
Immunofluorescence micrographs of transformed (31 C)
6m2 cells grown in the absence (left column) or presence (right
column) of 10r6 M dexamethasone (dex) for 24 h followed by
a temperature upshift to 39 C and treatment with 10~6 M dex
for the lengths of time indicated.
Vol 3 No. 8
MOL ENDO-1989
1284
adversely affect the cellular machinery required for hormone-dependent nuclear translocation of receptors. A
considerable proportion of 6m2 cells contained predominately nuclear receptors 12 h after a concurrent temperature upshift and hormone treatment (Fig. 5), although in separate experiments cytoplasmic staining
was observed in some cells at this time (not shown).
Given the rapid nuclear translocation of GR protein in
transformed 6m2 cells (Fig. 2), we might expect that
some desensitized receptors would be generated if
hormone is added at the onset of a temperature upshift,
particularly since the complete turnover of cytoplasmic
p859agmos in vivo requires greater than or equal to 4 h
at 39 C (32).
Dexamethasone Treatment of p859agmosTransformed 6m2 Cells Lead to Accentuated
Down-Regulation of GR Protein
Glucocorticoids down-regulate GR protein levels in various cell types (13-16). Since hormone responsiveness
can be sensitive to changes in receptor levels (40, 41),
ligand induced down-regulation of receptors could influence the ability of a given response to persist upon
prolonged hormone treatment. Receptor levels cannot
be precisely quantified from IIF analyses, nor can any
inference be made from these experiments concerning
the physical form of receptors. In fact, the IIF micrographs shown in previous figures were printed to optimally visualize GR protein staining and thus are not
indicative of relative GR protein levels. The BuGR2
monoclonal antibody, which recognizes an epitope at
or near the DNA-binding domain of the rat GR protein,
will react with proteolytic fragments of receptors in
immunoblotting assays (42). We therefore performed
Western blot analysis to determine whether, 1) desensitized GR protein in hormone-treated transformed 6m2
cells was intact and, 2) v-mos-mediated desensitization
was associated with down-regulation of GR protein. In
nontransformed 6m2 cells, GR protein levels declined
slightly after a 24-h hormone treatment (Fig. 6, Table
2). In contrast, GR protein levels were decreased considerably after dexamethasone treatment of transformed 6m2 cells. For example, receptor levels were
reduced to 54% of unstimulated levels after only a 4-h
hormone treatment (Fig. 6, Table 2). Ultimately, GR
protein levels were reduced to 15% of unstimulated
levels after 24 h of hormone treatment (Fig. 6, Table 2).
Importantly, despite the down-regulation of GR protein
in transformed 6m2 cells, receptors appeared intact as
no other forms of immunoreactive protein, other than
the intact 94-kDa receptor, were observed at any time
after hormone treatment. Desensitized GR protein in
transformed 6m2 cell does not appear to be distinguished electrophoretically from the major immunoreactive receptor species in unstimulated cells, nor from
immunoreactive receptors in nontransformed 6m2 cells.
In order to corroborate the down-regulation of GR
protein in transformed 6m2 cells, and provide a more
accurate measure of receptor levels, the in vivo hor-
M
20511697.466-
31°C(T)
45-
200-
0
4
12
24
36
Hours in Dex
Fig. 6. Dexamethasone (dex) Treatment of p85ga9"mos-Transformed 6m2 Cells Lead to Accentuated Down-Regulation of
GR Protein
6m2 cells grown at 31 C (Transformed, T) and 39 C (Nontransformed, NT) were treated with 10"6 M dex for the lengths
of time indicated. Western blot analysis was performed as
described in Materials and Methods. Each lane contained 200
Mg total protein.
mone binding activity of GR protein in transformed and
nontransformed 6m2 cells was determined. As shown
with whole cell hormone binding assays, GR protein
levels estimated from Western blot analyses of transformed 6m2 cells corresponded closely with specific
hormone binding activity (Table 2). For example, after
a 24-h dexamethasone treatment of transformed 6m2
cells, GR protein levels were reduced to approximately
15% and 25% of unstimulated levels, as quantified from
Western blots (Fig. 6, Table 2) and whole cell binding
assays (Table 2), respectively. Since desensitized GR
protein is predominately cytoplasmic in hormone
treated transformed 6m2 cells (Fig. 2), we also measured the in vivo hormone binding activity of cytosolic
GR protein in hormone-treated transformed 6m2 cells.
Coincident with the results from whole cell binding
assays, the reduction in receptor protein levels estimated from Western blots corresponded well with reductions of the in vivo hormone binding activity of
cytosolic receptors in transformed 6m2 cells (Table 2).
In nontransformed 6m2 cells, whole cell binding assays
revealed a modest down-regulation of GR protein after
hormone treatment. Although an accurate measure of
the specific hormone binding activity of desensitized
receptors requires more direct analyses, the results
described above demonstrate that desensitized receptors in v-mos-transformed cells retain some capacity to
bind hormone in vivo.
DISCUSSION
We have uncovered a novel mechanism through which
hormone insensitivity is acquired by NRK cells that are
v-mos Effects on GR
1285
Table 2. Hormone and Antibody Binding Activity of GR in Transformed (T) and Nontransformed (NT) 6m2 Cells
% Maximum Specific Binding Activity
NT (39 C)
Time in Dex (h)
0
0.15
1
4
12
24
T (31 C)
we
IMM
we
CYTO
IMM
ND
33.9 ±11.3
100
70.2 ± 6.7
47.7 ± 2.6
55.1 ± 5.7
100
ND
ND
68
81
80
ND
25.5 ± 0.6
100
67.2 ± 2
28.8 ± 9.7
26.5 ± 1.6
ND
21.8 ± 1
100
49.5 ±12.7
14.7 ± 5 . 5
11.7 ± 5 . 2
100
ND
ND
54
22
15
For in vivo hormone binding assays, transformed (T, 31 C) and nontransformed (NT, 39 C) 6m2 cells were incubated in the presence
of 10-25 nM 3H-dexamethasone (3H-Dex) for the lengths of time indicated, and specific binding (mean values ± SD) in whole cells
(WC) and crude cytosols (CYTO) determined as described in Materials and Methods. Nonspecific binding (typically <5% of specific
binding) was monitored by inclusion of a 200-fold excess of unlabeled dexamethasone in duplicate plates. Relative levels of
immunoreactive (IMM) GR were determined from Western blot analyses using total protein extracted from cells treated with 1 ^M
dexamethasone (Dex) for the lengths of time indicated. ND, Not determined.
transformed by v-mos oncoproteins. The oncoprotein
appears to be responsible for defects in both the nuclear/cytoplasmic partitioning of GR protein and the
reutilization of recycled receptors. Thus, dexamethasone treatment of v-mos-transformed cells leads to only
a transient association of GR protein with the nucleus.
Receptors that translocate to the nucleus upon hormone treatment are rapidly depleted from that compartment and subsequently redistributed to the cytoplasm. Furthermore, once desensitized, GR protein remains cytoplasmic, despite the continued presence of
hormone, and after the turnover of a thermo-labile vmos oncoprotein. This later observation demonstrates
that the presence of a functional v-mos oncoprotein,
although required to desensitize GR protein, is not
required to maintain receptors in this state. Thus, desensitized receptors appear to be trapped in the cytoplasm, and henceforth are incapable of being reutilized.
Does the Accumulation of Desensitized Receptors
in v-mos Transformed Cells Reflect Alterations in
the Processing of Recycled Cytoplasmic
Receptors?
v-mos affects not only the subcellular distribution of GR
protein, but also the extent of ligand induced downregulation of receptor. GR protein is down-regulated by
glucocorticoids to different extents in a variety of tissues and cultured cells (13-16). This down-regulation
reflects both decreased GR protein and mRNA levels
(14). Analogously, in transformed 6m2 cells the accentuated down-regulation of GR protein is also accompanied by an accentuated down-regulation of GR
mRNA levels (Hamilton, B. J., and D. DeFranco, unpublished). However, in striking contrast to our observations, down-regulation in other characterized systems
did not lead to alterations in the subcellular distribution
of GR protein (13,15, 43). In these cases the predominant nuclear localization of receptors in hormone
treated cells remained unchanged as receptor levels
were coordinately reduced from both nuclear and cy-
toplasmic compartments (13, 15, 43). Although GR
protein is down-regulated in transformed 6m2 cells,
desensitized receptors in v-mos-transformed cells are
distinguished by their inability to be retained in the
nucleus of hormone treated cells. Thus, v-mos effects
on the nuclear/cytoplasmic partitioning of hormonebound GR protein are superimposed upon agonist dependent down-regulation of receptors.
It has been postulated that nuclear GR protein can
redistribute to the cytoplasm via recycling pathways
that involve either a reversible cytoplasmic to nuclear
exchange of agonist-bound receptors (18) or a cyclic
pathway (17, 19) that implicates the existence of novel
intermediate recycled receptor forms. Oncoprotein-mediated defects in the proposed cyclic pathway would
be most compatible with the observed accumulation of
desensitized cytoplasmic GR protein in v-mos-transformed cells. Since hormone-dependent translocation
of GR protein into the nucleus is apparently normal in
v-mos-transformed cells, we assume that hormone
binding and subsequent activation of previously unstimulated receptors is unaffected by the presence of vmos. Thus, v-mos oncoproteins could effectively block
reutilization of recycled receptors without exerting any
effects upon the activation and nuclear transport of
previously unstimulated receptors, by distinguishing recycled from other cytoplasmic forms of receptor. Since
desensitized receptors appear to retain some capacity
to bind hormone in vivo, the inability to reutilize desensitized receptors may be due to some alteration in the
receptor recycling pathway subsequent to hormone
binding. Biochemical analyses of GR protein in v-mostransformed cells may establish the relationship between desensitized receptors and postulated recycled
receptors and also elucidate molecular mechanisms
involved in GR processing and recycling.
Since steroid receptors are known to be phosphoproteins (10), numerous studies have been directed
towards uncovering a physiological role for this modification. Although the activation of GR protein does not
apparently alter its phosphorylation state in vivo (44-
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MOL ENDO-1989
1286
46), it had been postulated based upon in vitro experiments that phosphorylation of receptors in the cytoplasm might be associated with their recycling (47).
Progesterone receptors have been shown to be phosphorylated in both cytoplasmic and nuclear compartments (48, 49), but there is no direct evidence that
these modifications alter any biological activity of progesterone receptors. It is tempting to speculate that vmos oncoproteins, cytoplasmic protein serine/threonine
kinases (28, 31), directly phosphorylate GRs, although
this appears unlikely given our inability to detect immunoreactive receptor forms with altered electrophoretic mobilities in hormone-treated transformed 6m2
cells. Since our present physical analyses of desensitized GR protein are by no means complete, direct
modification of GR protein by v-mos oncoproteins cannot be completely excluded at this time. However, it
also seems likely that desensitization could be due to
the stable association of recycled receptors in v-mostransformed cells with cytoplasmic inhibitory factors. If
these cytoplasmic factors in fact represent substrates
of p859ag"mos, they must be able to distinguish recycled
from unstimulated GR protein. Furthermore, the putative stable association of normal recycled receptors
with modified cytoplasmic factors would not require the
continuous presence of p859a9"mos as desensitization of
GR protein cannot be reversed after turnover of this
thermo-labile oncoprotein. Interestingly, cytoplasmic localization and inactivity of another enhancer binding
protein, NF-KB, has been shown to most likely result
from its stable association with cytoplasmic inhibitory
factors (50). In this case, activation of protein kinase C
in the appropriate cell types leads to rapid nuclear
translocation of NF-KB, and uncovering of its DNA-
binding activity (50). We currently do not know whether
desensitization of GR protein is reversible, since neither
the continued presence of steroid hormone nor turnover
of the p859agmos oncoprotein relieves the desensitization
of glucocorticoid receptors in transformed 6m2 cells.
(53). The properties of GR protein that we have observed in v-mos transformed 6m2 cells (i.e. desensitized and inefficiently retained in the nucleus) are strikingly analogous to properties associated with receptors
present in the G2 phase of the HeLa cell cycle. Curiously, a novel form of GR protein accumulates during
the G2 phase of the HeLa cell cycle (54). If desensitized
receptors in transformed 6m2 cells do in fact correspond to a G2 form of GR protein, a useful biochemical
marker would be provided for further analyses of v-mos
effects on the inappropriate expression of phenotypes
associated with G2-M phase transitions.
The fact that oncogene effects on steroid hormone
regulation can reflect an alteration in the nuclear/cytoplasmic partitioning of a steroid receptor protein suggests targets of oncoprotein action that could account
for many alterations in the normal regulation of cellular
growth and proliferation. Detailed analyses of the mechanisms that are responsible for oncoprotein effects on
the biological activities of a specific signal transducer
(i.e. steroid receptor protein), may provide insights into
the basis of oncogene effects on a variety of cellular
responses.
MATERIALS AND METHODS
Cells
6m2 (30) and 54-5A4 (37) rat cell lines (kindly provided by R.
B. Arlinghaus) were maintained at 31 C in McCoy's 5A medium
(GIBCO, Grand Island, NY) supplemented with 15% fetal bovine serum (GIBCO), while the NRK cell line was maintained
at 37 C in Dulbecco's Modified Eagles Medium (GIBCO) supplemented with 10% fetal bovine serum. For inductions performed at the nonpermissive temperature, cells were shifted
to 39 C and maintained for at least 16 h before any treatment.
Dexamethasone and CdCI2 (Sigma, St. Louis, MO) were added
to approximately 60-80% confluent cell cultures in normal
growth medium to attain a final concentration of 10~6 M.
RNA Isolation and Analysis
Is the Accumulation of Desensitized GR Protein a
Biochemical Marker of v-mos Mediated Alterations
in Cell Cycle Progression?
Based upon the function of c-mos in meiotic maturation,
Vande and co-workers (51) have postulated a role for
c-mos in cell cycle progression, particularly in activating
some G2-M phase transition events. Furthermore, they
also suggest that v-mos mediated transformation may
reflect the inappropriate expression of cellular phenotypes normally associated with G2-M phase transitions
(51). Interestingly, GR protein levels have been shown
to fluctuate during the HeLa cycle (52). During most
phases of the HeLa cell cycle nuclear and cytoplasmic
receptors change coordinately in the continued presence of hormone, with the noteable exception of the
G2 phase, where nuclear receptor levels disproportionately decline (52, 53). In addition, various glucocorticoid
responsive markers become insensitive to hormone
treatment as HeLa cells progress through the G2 phase
Isolation of RNA, Northern blot analyses, and nucleic acid
hybridizations were performed as described previously (32).
P-Labeled DNA probes (pMT-1 and pBR/3actin) were prepared for hybridization using nick translation as described
previously (32).
32
Western Blot Analysis
A cell-free lysate was prepared from four confluent 100-mM
plates of cells by sonication in TEGN150 buffer (10 ITIM TrisCl, pH 7.5, 1 mM EDTA, 150 mM NaCI, 1 rriM /3-mercaptoethanol, and 10% (vol/vol) glycerol) as described previously (55).
Two hundred micrograms of total protein from cell free lysates
were applied per lane, separated by electrophoresis through
10% sodium dodecyl sulfate polyacrylamide gels, and electroblotted onto nitrocellulose membranes. Duplicate gels were
stained to visualize total protein and ensure that equivalent
amounts of protein were loaded per lane. The subsequent
processing of filters for detection of receptor protein was
performed using a Protoblot AP System (Promega, Madison,
Wl) essentially as described previously (42). The BuGR2 monoclonal antibody against rat GR protein (kindly provided by R.
Harrison; 36), was used at a 1:20 dilution and incubated for 1
1287
v-mos Effects on GR
h at room temperature. Laser densitometry of photographed
blots was used to quantify relative GR protein levels.
and by a Basil O'Connor Research Award from the March of
Dimes Foundation.
Immunofluorescence Assays
IF was carried out essentially as described previously (12)
with the following exceptions. In all experiments, cells were
shifted to DMEM lacking phenol red (GIBCO) supplemented
with 10% steroid stripped fetal bovine serum, and grown for
16 h before any hormone treatment and fixation. Cells were
fixed by the addition of cold (-20 C) methanol, followed by
incubation at room temperature for 5 min. The BuGR2 monoclonal antibody was added undiluted, and incubated for 1-2 h
at 37 C. The second antibody, FITC-conjugated anti-mouse
immunoglobulin G (Pharmacia, Piscataway, NJ), was added at
a 1:50 dilution for 1 - 2 h at 37 C.
In Vivo Hormone Binding Assays
Two 100-rriM plates of cells, approximately 60-80% confluent,
were shifted to medium supplemented with steroid stripped
serum and grown for at least 16 h before any hormone
treatment. 3H-Dexamethasone (New England Nuclear, Boston,
MA >40 Ci/mmol) was added to a final concentration of 1025 nM in the presence and absence of a 200-fold excess of
unlabeled dexamethasone, and cells were incubated for the
lengths of time indicated in Table 2. It should be noted that at
early time points (0 and 15 min), equilibrium binding may not
have been attained. For whole cell binding assays, an impermeable agent, 14C-sucrose (New England Nuclear, 0.67 Ci/
mmol) was added to monitor the efficiency of washing radiolabeled medium from cells (typically, contamination of cell
pellets with radiolabeled medium was <0.01%). After extensive washing with ice-cold PBS, cells were harvested, and
then either counted directly in scintillation fluid for whole cell
binding assays (after removing a sample for protein determination), or lysed using a variation of a freeze-thaw procedure
described previously for determination of cytosolic binding
(45). The lysis buffer contained 20 ITIM Tris-CI, pH 7.2, 50 mM
NaCI, 1 mM EDTA, 5 mM dithiothreitol, 20 mM Na2MoO4, and
10% (vol/vol) glycerol. This lysis procedure typically yielded
approximately 20-50 ng total cytoplasmic protein/100 mM
plate of cells. After two cycles of freeze-thawing, the cell lysate
was centrifuged at 1500 x g for 10 min at 4 C, and the quantity
of 3H-dexamethasone bound receptor in the resultant supernatant determined using a charcoal absorption assay (55).
Nonspecific binding of hormone to cytosolic receptor, monitored by the inclusion of excess unlabeled dexamethasone
(see above) was typically less than 5% of specific binding.
Maximum specific binding was usually greater than or equal
to 100,000 cpm/mg total protein.
Acknowledgments
We are deeply indebted to Chuck Walsh for his advise on
immunofluorescence and the use of his Zeiss Universal epifluorescence microscope. We also appreciate the kind gifts of
BuGR2 monoclonal antibody from Bob Harrison, and 6m2 and
54-5A4 cells from Ralph Arlinghaus. Eileen Lafer and Mike
Garabedian are thanked for their advise on Western blotting,
and Bob Harrison and Didi Robins for critical reading of the
manuscript and helpful discussions. Finally, Sandra Rusconi is
thanked for sharing unpublished observations and valuable
discussions.
Received April 26, 1989. Revision received May 18, 1989.
Accepted May 18, 1989.
Address requests for reprints to: Dr. Donald DeFranco,
University of Pittsburgh, Department of Biological Sciences,
Pittsburgh, Pennsylvania 15260.
This work was supported by Grant CA-43037 from the NIH
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