Endocrinol Japon
1992, 39 (5), 477-483
In Vitro Monitoring Activation by the Ligands and Specific DNA-Binding of the Glucocorticoid Receptors HIROTOSHITANAKA, ETSUSHIFUKAWA, HUMINORIHIRANO, YuIcHI MAKINO, AKIRAAOKI, YUKOMORIKAWA, YuMI TAKIYAMA, TAKAKOTANI ANDISAOMAKINO SecondDepartmentofInternal Medicine, AsahikawaMedicalCollege,Asahikawa078, Japan
Abstract. The glucocorticoid receptor is a member of the steroid and thyroid hormone receptor superfamily and acts as a ligand-activated transcription factor. To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits glucocorticoid-induced activationof the latent cytosolic glucocorticoid receptor to an active DNA-binding species. We demonstrate here that cytosol from a rat hepatoma cell, M1.19, contains glucocorticoid receptor-specific immunoreactivities and target DNA-binding activities. Moreover, specific DNA-binding activities of M1.19 cytosol were dose-dependently induced by dexamethasone treatment, and linearly correlated with the hormonal induction of chloramphenicol acetyltransferase activity at the corresponding concentrations. These results indicate that the cytosolic glucocorticoid receptor could be converted in a DNA-binding form under cell-free conditions and the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex. Key words: DNA-binding mammalian cell.
protein, Chloramphenicol
GLUCOCORTICOIDS exert their regulatory effects by binding to the glucocorticoid receptor (GR), one of the superfamily and a modulator of gene
of nuclear receptors transcription, either
positively or negatively [1]. On binding hormone, GR undergoes transformation and translocates to the nucleus, and there binds to the target DNA sequence termed glucocorticoid-response element (GRE, reviewed in [2-4]). However, the mechanism of activation to a functional species is not yet well understood, and a number of different regulatory mechanisms have been postulated to control this process. These models include, among others, intramolecular changes in receptor conReceived: April 28, 1992 Accepted: July 17, 1992 Correspondence to: Dr. Hirotoshi TANAKA, Second Department of Internal Medicine, Asahikawa Medical College, 4-5-3-11 Nishikagura, Asahikawa 078, Japan.
acetyltransferase, Transient transfection, Cultured (EndocrinolJapon 39: 477-483, 1992)
formation or intermolecular interactions with distinct proteins (reviewed in [2-5]). For instance, hormone has been shown to induce dimerization of GR, a critical event which determines high affinity interaction with GRE [6-8]. More importantly, GR has been shown to be associated with the 90 kDa heat shock protein, hsp90 (reviewed in [2-5]). Furthermore, hsp90 appears to repress the DNA-binding activity of GR, since interaction of the receptor with target DNA sequences is observed only following dissociation of hsp90 (reviewed in [2-5]). In particular, the role of ligands in GR-DNA interaction has been strongly debated, given that in vivo and in vitro studies have yielded conflicting results [9-12]. The most likely explanation for this controversy is the fact that non activated, heteromeric GR complexes represent a very labile complex which readily breaks down in vitro.
478
TANAKA
et al.
the Prot Blot Immunoblotting System (Promega) using an alkaline phosphatase-conjugated second anti-mouse goat antibody as described [14].
However, under conditions exposing the heteromeric GR complex to as few in vitro manipulations as possible, Denis et al have shown that hormone is required for activation of the DNAbinding activity of GR and concomitant release of hsp90 [13]. Using their procedure, we have further developed an in vitro activation system for GR using crude cytosol from rat hepatoma cell M1.19, in which we can monitor the dose-dependent activation process of GR with regard to its specific DNA-binding activities. Moreover, we show that the acquisition of DNA-binding activity correlated with their potency in inducing target gene activation.
DNase I footprint experiment A
plasmid
sites
of
pLS5'139
the
mouse
promoter
DNA
upstream
half
185-bp
[a-32P]
of
was
Methods
in
culture
500
cia),
rat
hepatoma
Dulbecco's
cells
modified
mented
with
pretreated
Eagle's
10%
with
were
fetal
medium
calf
at
which
charcoal
37•Ž
in
a
in
I
phere.
of
Cells
cytosol
were
cultured
confluence
and
described
before
and
EDTA,
in
10mM
at
supernatant
was
The
concentration
Protein
turer's
Tris
for
used
as
a
HC1,
45
Kit
pH
min
at
in
to
for
min,
gel.
The
gels
One
hundred were
micrograms
of on
sulfate-polyacrylamide onto
a
identified mouse Gustafsson,
gel
nitrocellulose
Richmond,
a
CA). by
means
antibodies Karolinska
and
Electrophoresis
GR of
([14],
protein
blotted
and
Dr.
sulfate,
of carrier incubated
20 yeastu 37•Ž
at
then
analy-
polyacrylamide a
3MM
Hyper-film
paper
and
overnight
at
shift
assay
(EMSA)
the
complexes
samples gel
on
[16].
The
encompassing
the
ferase
gene
as
(TAT)
were
a low
ionic
identified strength
following
GRE [2]
in
run-
oligonucleotides
the
were
by
polyacryl-
tyrosine synthesized
aminotransand
used
probe:
5'-TCGACCTCAGAACATCCTGTTCTAGC-
GR
Jan-Ake
visualized
buffer
GG-3',
were
anti-rat by
reaction
a stop
ethanol,
on
was
The
dodecyl ug
protein
5'-CGAGTAGCTAGAACAGGATGATCTGA-
protocol, bands
monoclonal
per
dodecyl
(Bio-Rad
provided Institute)
protein
sodium
the
urea-6%
dried
at DNase
digestion
of
sodium
mobility
Protein-DNA
manufac-
electrically
filter The
cytosol
7%
incubated
ice.
100ul
Amersham
10%
a
analysis
separated
on
mM
and
upon
min
7.9),
2.5
-
amide
lane
(pH
the
ning
immunoblot
Hepes
then
8M
per-
(40,000
70•Ž.
protocol.
Western
was
probe
was
with
were
on
1 uM subse-
freshly-diluted
K) with 5 was further
denaturing
a
the
spermidine,
adding
precipitated
a
end
(Pharma-
5 mM
of
2
0.2%
was
Cytosol
for
of
added,
by
the
of
dithiothreitol,
5ul
for
and
Klenow
reaction
fmoles
depending
EDTA,
on
min
mixture
and
proteinase The sample
exposed
study. with
the
mM
30
mM
reaction
g/ml tRNA.
the
4•Ž,
this
1
mM
of
(10
the
presence
in
10
proceed
terminated
30
5
was
was
using
mM
KC1,
ng/ml
to
pLS5'139
binding
cytosol
min,
(1-100
zed
7.4, 2
determined
according
as lyzed
and
cytosol was
near
were
glycerol
200,000•~g
Assay
cells
centrifugation
homogenate
Pierce
at essentially
brief,
(vol/vol)
After
protein
harvested prepared
In
10%
dithiothreitol.
was
[14].
homogenized
mM
and
cytosol
mM
20
1.4
experiments,
poly(dI-dC)-poly(dI-dC)
concentration)
atmos-
for
0.25
The for
allowed
Preparation
of of
60
25•Ž
peni-
CO2
25•Ž
with
EDTA,
glycerol.
was
and
10%
mM
MgCl2,
supple-
serum
dextran-coated
cillin/streptomycin
cultured
ng
0-200 ug
2.5 M1.19
the
A
100ul
(MMTV)
(Promega).
in
experiments.
cpm), Cell
at
formed
of
polymerase
pre-incubated
1.3,
For
dATP
DNA
GR-binding virus
as [15]).
fragment
with
fragment
quent
and
1.5
SstI-EcoRI
labeled
three tumor
[designated of
dexamethasone
Materials
contains
mammary
TACTCGAGCT-3' These
oligonucleotides
labeled by
treated
with or
nontreated
were
[a-32P]dATP. cytosol
annealed
and
end
Dexamethasone(0-10 ug
protein
per
IN
reaction)
was
endlabeled cpm)
in
the
with
5
or
composition
of
which
carried
out
analyzed
on
was
samples
were
native
gel.
DNA
The
gels
of
in
a
were
479
or buffer above.
for
3.5%
GR
32P-
specific
described ice
OF
(20,000
a binding
is on
ACTIVATION
of
DNA
presence
competitor
Incubation
frnoles
probe
absence
non-specific the
incubated
double-stranded
VITRO
20
min
and
polyacrylamide
dried
and
autoradiog-
raphed.
DNA
transfection
(CAT)
and
chloramphenicol
The
reporter
driven
plasmid
under
the
were
from
into
lipofection viously dish
twice
cells as
In
with
brief,
2
with
ml
After
cells
of 12
replaced
10
h
of
with
presence
of
prepared
1 ƒÊg
Lipofectin
to the
for
BKL)
the
culture.
medium
fetal
calf
CAT
Fig. 1.
was medium
serum
for
18
Cellular
assayed
pCH110)
Eagle's
continued
60
Plasmid
of
(GIBCO
added
10%
a
washed
BRL). or
dexamethasone.
and
on and
modified
further
the pre-
cultured
transfection,
with was
were
and
Dulbecco's
supplemented culture
by
described
confluence
,ƒÊg of
trans-
performed
(GIBCO
OptiMEM,
pCH110
Transient
pMSGCAT
is
promoter,
plasmid
was
until
of
which
MMTV
essentially
OptiMEM
(5 ƒÊg mixed
in
M1.19
(Corning)
cocktail
the
Pharmacia.
procedure [17].
mm
of
expression
obtained
fection
pMSGCAT,
control
and ƒÀ-galactosidase
was
acetyltransferase
assays
and
h
in
extracts
enzyme
the were
activity
as
described [18].
between -188 and —-166,-128 and further downstream of the MMTV probe DNA against DNase I digestion (Fig. 2). The localization of
Results CR-specific vities
of
To
immunoreactivities cytosol
detect
from
the
and
a
rat
DNA-binding
hepatoma
GR-specific
western
immunoblotting
monoclonal
anti-GR
antibodies
the
presence
immunoreactivity molecular
in weight
ponded
to
the
confirmation carried the
out MMTV
1 ƒÊM
a
M1.19
DNase promoter M1.19
dexamethasone,
as cell
the
calculated and
[2,14].
footprint
For GR,
corresfurther we
experiment
probe. cytosol, protected
1
GR-specific
94-kDa
of
the
Figure
single
cytosol, was
presence I
with
a
reports
the
M1.19
we
[14].
of
which
previous of
experiment, with
of
acti-
line
immunoreactivity,
performed
demonstrates
cell
In after
next with
the
present treatment
the
regions
Western immunoblotting of cytosol isolated from a rat hepatoma cell line M 1.19. One-hundred micrograms of cytosol protein were analyzed on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were electrically blotted onto a nitrocellulose filter membrane. Specific interaction between GR protein and anti-rat GR mouse monoclonal antibody was visualized by means of the horse-radish peroxydase-conjugated anti-mouse goat antibody. Molecular weight was determined by the marker run in parallel.
protected regions was shown to be functional with regard to glucocorticoid-inducible transcriptional enhancement (reviewed in [2-4]), and essentially identical to the results from previous experiments in which purified GR were used [15], except for failure to protect the region between -159 and 135. However, similar results to ours were reported by Willmann and Beato with cytosol as a source of GR [10]. Accordingly, these results indicated that cytosol from M1.19 cells contains GR-specific immunoreactivities and glucocorticoid-inducible sequence specific DNA-binding activities.
480
TANAKA
et at.
In vitro activation of cytosol by dexamethasone and monitoring specific DNA-binding activities of GR
concerning GR
the
in
vitro
role
reconstituted
Previous reports have given conflicting results
in
ment
with
from
M1.19
vitro
were
from
these
binding
cytosol
competed
the
indicated
molar
TAT
GRE
encompassing
not
interfere
3B,
see
with
also
legend),
could
be
complex
After gical
concentrations
also
see
incubated on
with
EMSA
incubated
with
probe
urea-polyacrylamide protein G-sequence of I
the
of
gel. cytosol, ladders
downstream
treatment
DNA.
are
M1.19 cytosol preparation)
After
partial
Lane
1,
respectively. run in parallel. footprint. boxed.
(B) Numbers
100 ƒÊg
of
the
using
serum
depict regions
nuleotide
albumin
nucleotide are indicated of
the
MMTV
positions
MMTV
was
DNase
the
treated
I,
absence
samples for
were
control;
positions by a line promoter. relative
lanes
determined except for
the
nM, was
analyzed
amount
hormone in
as
a
the regions
transcription
concen-
(See and
denaturing
50, by
100,
the lower
cyto(lane
probe
on
2-4,
of
and
dexamethasone
analyzed
Protected to
DNA
1 ƒÊm
(5-20
GRE
increased
in
cytosol and
of
an GRE.
physiolo-
vitro,
a trace
promoter with
EMSA
and
nearly
TAT
for did (Fig.
of
probe
the
[20]
our
GR
in
GRE
formation
32P-labeled
that
Although
in
complex
3C)
between
observed
Cytosol by
bovine
sequences
depict
2),
digestion
Numbers Protected DNA
was
SRE
observation
with
for of
sites
or
dexamethasone
TAT
formation
sol
. (A)
the
specific
formation,
cytosol
the this
excess
between
Fig.
(Fig.3C).
complex
with probe
to
be
[19]
for
of
legend
to
indicating
formation of
in
Moreover,
complex
all
unlabeled
binding
relevant
treatment
of present
molar
AP-1
the
(Fig.
between
dose-dependently
shown
factors
transcription
,ƒÊM
DNA-
probe
formed
100-times
the
1 of
GRE
3-6).
oligonucleotides
vitro
experiment Methods" for
a
Cytosol
class
was
was
since
of activi-
32P-labeled
excess
lanes
formation GRE,
cytosol
probe.
single
oligonucleotides 3A,
treat-
of
with
TAT
and
system
I footprint and
the
when
(Fig.
with a
complexes
GRE
TAT
DNase "Materials
a
of
after
presence
incubation
with
amounts
the
we
binding
as
after
activities The
complex
in
of
issue,
GR
EMSA
revealed
reaction
2.
GRE-specific
oligonucleotide
dexamethasone,
Fig.
incubation
the
in
activation this
preparation
and
cells,
the
of
the
monitored
GRE
for address
activation
After
cells
TAT
3A).
ligands To
ligands.
dexamethasone, ties
of
[10-12].
200 ƒÊg
A/G and boundary
from
DNase
start
site.
a
Fig.
3.
Electrophoresis
mobility
was
with
pretreated
cytosol (B)
and
0,
2,
Competition
probe in the oligonucleotides, AP-1;
the
5,
VITRO
ACT1VA
assay
using
32P-labeled
1ƒÊM.1\4 dexamethasone 20,
and
100-fold
experiments. absence
(lane respectively.
at molar
Cytosol
was
10,
[20]. 20
presence
oligonucleotides,
nM
of of
(C)
I,
dexamethasone 20 C1,
nM
probe at
of
protein-DNA
only; 25•Ž
of
pretreated
for
15
dexamethasone complex
tration-dependent fashion when cytosol was treated with increasing concentrations of dexamethasone in vitro (lanes 3-5). To verify the physiological significance of the activation process of GR as represented above, we measured the transcriptional effects of dexamethasone at the corresponding concentrations of dexamethasone using the MMTV-driven CAT reporter plasmid pMSGCAT. After transient transfection of the plasmids, M1.19 cells were treated with the indicated concentrations of de-
30
GR
GRE min.
481
oligonucleotides Lane
unlabeled with
100-times DNA
lanes
OF
TAT for
excess
1) or presence of The non-coding
Lane
HON
25•Ž
5'-CTAGIGATGAGICAGCCGGATC-3'
CTGCGTC-3' 0,
5,
shift
IN
TAT 1ƒÊM
molar sequences
1,
as a probe,
probe
GRE
only;
the AP-1 competitor
incubated 2) or SRE oligonucleotides
SRE;
5'-CTAGAGGATGTCCATATTAGGACAT-
10ƒÊg
of cytosol
and formed;
respectively; 100-fold F,
lane molar
free
was 6,
preincubated 10ƒÊg
excess
of of
of
respectively.
(lane
[19],
Cytosol 10 ƒÊg
then
2-5, min,
(A) 2-6,
oligonucleotides,
dexamethasone,
excess of of these
lanes
in cytosol unlabeled
the
with (lane
presence
the 3) are:
of
preincubated TAT
in GRE
probe.
xamethasone, and the CAT activity of the cellular extracts was determined. Treatment with dexamethasone induced the CAT expression in a concentration dependent fashion (Fig. 4). Together with results from EMSA, we may conclude that, at physiological concentrations of agonistic ligands, GR is activated and allowed to bind its cognate sequences of target genes, and the amounts of activated fraction of GR correlate with the magnitude of subsequent transcriptional effects.
482
Fig.
TANAKA
4.
Expression cells.
of The
galactosidase control for the
lated
cells
concentrations of forms
activity plasmid
were of
rat
hepatoma
pCH110 were
and ƒÀ(as internal transfected by
"Materials in
the
dexamethasone
chloramphenicol
M1.19
pMSGCAT
(see cultured
[14C]-chloramphenicol Of
in
plasmid efficiency)
procedure
and
positions
CAT
expression transfection
lipofectin
Methods") the
the
reporter
and
presence
indicated. (CM) (ACM)
and are
of The acety-
shown.
Discussion We demonstrate here that M1.19 cell cytosol contains cryptic GRE-binding activities that can be dose-dependently induced in, vitro by treatment with dexamethasone. Moreover, these induced DNA-binding activities correlated with in vivo hormonal induction of CAT activity when cells were treated with corresponding concentrations of dexamethasone. In several instances, regulation of the inducible gene expression has been shown to involve posttranslational modification of a pre-existing latent transcription factor to an active form which specifically hinds to regulatory DNA elements in target genes mode
(reviewed in [21]). Here, we show a similar of action of GR which requires ligand-
et al.
dependent conversion in vivo from a non DNAbinding to a DNA-binding form, which we can faithfully mimic in vitro. Although previous studies have dealt with the kinetics between GR and target DNA sequences using purified or expressed fraction as a source of GR [6, 7], it has been very difficult to establish an in vitro activation system for cytosolic GR. One possible explanation for this is that GR protein is labile and susceptible to various in vitro manipulations. Indeed, exposing cytosol to as few in vitro manipulation as possible [13], our system presented here enabled us to monitor activation process of cytosolic GR, since receptor activation is dose-dependently represented by the acquisition of GRE-binding activity. Moreover, we show that the induction of the specific DNAbinding activity in EMSA, reflected the transcriptional potential of the ligands at the corresponding concentrations in vivo. These results therefore favor the idea that receptor activation represents a key regulatory event controlling receptor function. This linear relationship between in vitro activation of cryptic receptor in cytosol and in vivo receptor function strongly argues that hormone effects are likely to be quantitatively regulated at the level of receptor activation and subsequent DNA-binding. Of course, for further confirmation of this, other intracellular events after ligand binding [i.e., nuclear translocation] should also be examined.
Acknowledgments The authors wish to thank Dr. Jan-Ake Gustafsson for generously providing anti-GR antibodies and plasmids. We also thank to Hiromi Oba and Sachiko Iwakami for secretarial assistance. This work was partly supported by the grants to H.T. from Hokkaido Prefecture and the Japan Rheumatism Foundation.
References 1. 2. 3.
Evans RM (1988) The steroid and thyroid receptor superfamily. Science 241: 889-895. Beato M (1989) Gene regulation by steroid hormones. Cell 56: 335-344. Muller M, Renkawitz R (1991) The glucocorticoid receptor. Biochim Biophys Acta 1088: 171-182.
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M (1991) Structure
C (1991) Biological
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role and regulation
of
IN
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7.
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11.
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13.
VITRO
ACTIVATION
the universally conserved heat shock proteins. J Biol Chem 266: 24233-24236. Tsai SY, Carlstedt-Duke J, Weigel N, Dahlmen K, Gustafsson J-A, Tsai M-J, O'Malley B (1988) Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell 55: 361-369. Eriksson P, Wrdnge O (1990) Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties. J Biol Chem 265: 3535-3542. Cairns W, Cairns C, Pongratz I, Poellinger L, Okret S (1991) Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element. J Biol Chem 266: 11221-11226. Becker PB, Gloss B, Schmid W, Strahle U, Schatz G (1986) In vivo protein-DNA interactions in a glucocorticoid response element require the presence of hormone. Nature 324: 686-688. Willmann T, Beato M (1986) Steroid free glucocorticoid receptor binds specifically to mouse mammary tumor virus DNA. Nature 324: 688-691. Schauer MG, Chalepakis G, Willmann T, Beato M (1989) Binding of hormone accelerates the kinetics of glucocorticoid and progesterone receptor binding to DNA. Proc Natl Acad Sci USA 86: 1123-1127. Sanchez ER (1992) Heat shock induces translocation to the nucleus of the unliganded glucocorticoid receptor. J Biol Chem 267: 17-20. Denis M, Poellinger L, Wikstrom A-C, Gustafsson J-A (1988) Requirement of hormone for thermal conversion of the glucocorticoid receptor to a
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DNA-binding state. Nature 333: 686-688. 14. Dong Y, Poellinger L, Gustafsson J-A, Okret, S (1988) Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranscriptional mechanisms. Mol Endocrinol 2: 1256-1264. 15. Wrange O, Carlstedt-Duke J, Gustafsson J-A (1986) Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA. J Biol Chem 261: 11770-11778. 16. Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucl Acid Res 9: 6505-6525. 17. Tanaka H, Dong Y, Li Q, Okret S, Gustafsson J-A (1991) Identification and characterization of a cis-acting element that interferes with glucocorticoid-inducible activation of the mouse mammary tumor virus promoter. Proc Natl Acad Sci USA 88: 5393-5397. 18. Gorman CM, Moffat LF, Howard BH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 2: 1044-1051. 19. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin, M (1987) Phorbol ester-inducible genes contain a common cis element recognized by a TPAmodulated trans-acting factor. Cell 49: 729-739. 20. Treisman R (1986) Identification of a proteinbinding site that mediates transcription of the c-fos gene to serum factors. Cell 46: 567-574.
1992, 39 (5), 477-483
In Vitro Monitoring Activation by the Ligands and Specific DNA-Binding of the Glucocorticoid Receptors HIROTOSHITANAKA, ETSUSHIFUKAWA, HUMINORIHIRANO, YuIcHI MAKINO, AKIRAAOKI, YUKOMORIKAWA, YuMI TAKIYAMA, TAKAKOTANI ANDISAOMAKINO SecondDepartmentofInternal Medicine, AsahikawaMedicalCollege,Asahikawa078, Japan
Abstract. The glucocorticoid receptor is a member of the steroid and thyroid hormone receptor superfamily and acts as a ligand-activated transcription factor. To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits glucocorticoid-induced activationof the latent cytosolic glucocorticoid receptor to an active DNA-binding species. We demonstrate here that cytosol from a rat hepatoma cell, M1.19, contains glucocorticoid receptor-specific immunoreactivities and target DNA-binding activities. Moreover, specific DNA-binding activities of M1.19 cytosol were dose-dependently induced by dexamethasone treatment, and linearly correlated with the hormonal induction of chloramphenicol acetyltransferase activity at the corresponding concentrations. These results indicate that the cytosolic glucocorticoid receptor could be converted in a DNA-binding form under cell-free conditions and the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex. Key words: DNA-binding mammalian cell.
protein, Chloramphenicol
GLUCOCORTICOIDS exert their regulatory effects by binding to the glucocorticoid receptor (GR), one of the superfamily and a modulator of gene
of nuclear receptors transcription, either
positively or negatively [1]. On binding hormone, GR undergoes transformation and translocates to the nucleus, and there binds to the target DNA sequence termed glucocorticoid-response element (GRE, reviewed in [2-4]). However, the mechanism of activation to a functional species is not yet well understood, and a number of different regulatory mechanisms have been postulated to control this process. These models include, among others, intramolecular changes in receptor conReceived: April 28, 1992 Accepted: July 17, 1992 Correspondence to: Dr. Hirotoshi TANAKA, Second Department of Internal Medicine, Asahikawa Medical College, 4-5-3-11 Nishikagura, Asahikawa 078, Japan.
acetyltransferase, Transient transfection, Cultured (EndocrinolJapon 39: 477-483, 1992)
formation or intermolecular interactions with distinct proteins (reviewed in [2-5]). For instance, hormone has been shown to induce dimerization of GR, a critical event which determines high affinity interaction with GRE [6-8]. More importantly, GR has been shown to be associated with the 90 kDa heat shock protein, hsp90 (reviewed in [2-5]). Furthermore, hsp90 appears to repress the DNA-binding activity of GR, since interaction of the receptor with target DNA sequences is observed only following dissociation of hsp90 (reviewed in [2-5]). In particular, the role of ligands in GR-DNA interaction has been strongly debated, given that in vivo and in vitro studies have yielded conflicting results [9-12]. The most likely explanation for this controversy is the fact that non activated, heteromeric GR complexes represent a very labile complex which readily breaks down in vitro.
478
TANAKA
et al.
the Prot Blot Immunoblotting System (Promega) using an alkaline phosphatase-conjugated second anti-mouse goat antibody as described [14].
However, under conditions exposing the heteromeric GR complex to as few in vitro manipulations as possible, Denis et al have shown that hormone is required for activation of the DNAbinding activity of GR and concomitant release of hsp90 [13]. Using their procedure, we have further developed an in vitro activation system for GR using crude cytosol from rat hepatoma cell M1.19, in which we can monitor the dose-dependent activation process of GR with regard to its specific DNA-binding activities. Moreover, we show that the acquisition of DNA-binding activity correlated with their potency in inducing target gene activation.
DNase I footprint experiment A
plasmid
sites
of
pLS5'139
the
mouse
promoter
DNA
upstream
half
185-bp
[a-32P]
of
was
Methods
in
culture
500
cia),
rat
hepatoma
Dulbecco's
cells
modified
mented
with
pretreated
Eagle's
10%
with
were
fetal
medium
calf
at
which
charcoal
37•Ž
in
a
in
I
phere.
of
Cells
cytosol
were
cultured
confluence
and
described
before
and
EDTA,
in
10mM
at
supernatant
was
The
concentration
Protein
turer's
Tris
for
used
as
a
HC1,
45
Kit
pH
min
at
in
to
for
min,
gel.
The
gels
One
hundred were
micrograms
of on
sulfate-polyacrylamide onto
a
identified mouse Gustafsson,
gel
nitrocellulose
Richmond,
a
CA). by
means
antibodies Karolinska
and
Electrophoresis
GR of
([14],
protein
blotted
and
Dr.
sulfate,
of carrier incubated
20 yeastu 37•Ž
at
then
analy-
polyacrylamide a
3MM
Hyper-film
paper
and
overnight
at
shift
assay
(EMSA)
the
complexes
samples gel
on
[16].
The
encompassing
the
ferase
gene
as
(TAT)
were
a low
ionic
identified strength
following
GRE [2]
in
run-
oligonucleotides
the
were
by
polyacryl-
tyrosine synthesized
aminotransand
used
probe:
5'-TCGACCTCAGAACATCCTGTTCTAGC-
GR
Jan-Ake
visualized
buffer
GG-3',
were
anti-rat by
reaction
a stop
ethanol,
on
was
The
dodecyl ug
protein
5'-CGAGTAGCTAGAACAGGATGATCTGA-
protocol, bands
monoclonal
per
dodecyl
(Bio-Rad
provided Institute)
protein
sodium
the
urea-6%
dried
at DNase
digestion
of
sodium
mobility
Protein-DNA
manufac-
electrically
filter The
cytosol
7%
incubated
ice.
100ul
Amersham
10%
a
analysis
separated
on
mM
and
upon
min
7.9),
2.5
-
amide
lane
(pH
the
ning
immunoblot
Hepes
then
8M
per-
(40,000
70•Ž.
protocol.
Western
was
probe
was
with
were
on
1 uM subse-
freshly-diluted
K) with 5 was further
denaturing
a
the
spermidine,
adding
precipitated
a
end
(Pharma-
5 mM
of
2
0.2%
was
Cytosol
for
of
added,
by
the
of
dithiothreitol,
5ul
for
and
Klenow
reaction
fmoles
depending
EDTA,
on
min
mixture
and
proteinase The sample
exposed
study. with
the
mM
30
mM
reaction
g/ml tRNA.
the
4•Ž,
this
1
mM
of
(10
the
presence
in
10
proceed
terminated
30
5
was
was
using
mM
KC1,
ng/ml
to
pLS5'139
binding
cytosol
min,
(1-100
zed
7.4, 2
determined
according
as lyzed
and
cytosol was
near
were
glycerol
200,000•~g
Assay
cells
centrifugation
homogenate
Pierce
at essentially
brief,
(vol/vol)
After
protein
harvested prepared
In
10%
dithiothreitol.
was
[14].
homogenized
mM
and
cytosol
mM
20
1.4
experiments,
poly(dI-dC)-poly(dI-dC)
concentration)
atmos-
for
0.25
The for
allowed
Preparation
of of
60
25•Ž
peni-
CO2
25•Ž
with
EDTA,
glycerol.
was
and
10%
mM
MgCl2,
supple-
serum
dextran-coated
cillin/streptomycin
cultured
ng
0-200 ug
2.5 M1.19
the
A
100ul
(MMTV)
(Promega).
in
experiments.
cpm), Cell
at
formed
of
polymerase
pre-incubated
1.3,
For
dATP
DNA
GR-binding virus
as [15]).
fragment
with
fragment
quent
and
1.5
SstI-EcoRI
labeled
three tumor
[designated of
dexamethasone
Materials
contains
mammary
TACTCGAGCT-3' These
oligonucleotides
labeled by
treated
with or
nontreated
were
[a-32P]dATP. cytosol
annealed
and
end
Dexamethasone(0-10 ug
protein
per
IN
reaction)
was
endlabeled cpm)
in
the
with
5
or
composition
of
which
carried
out
analyzed
on
was
samples
were
native
gel.
DNA
The
gels
of
in
a
were
479
or buffer above.
for
3.5%
GR
32P-
specific
described ice
OF
(20,000
a binding
is on
ACTIVATION
of
DNA
presence
competitor
Incubation
frnoles
probe
absence
non-specific the
incubated
double-stranded
VITRO
20
min
and
polyacrylamide
dried
and
autoradiog-
raphed.
DNA
transfection
(CAT)
and
chloramphenicol
The
reporter
driven
plasmid
under
the
were
from
into
lipofection viously dish
twice
cells as
In
with
brief,
2
with
ml
After
cells
of 12
replaced
10
h
of
with
presence
of
prepared
1 ƒÊg
Lipofectin
to the
for
BKL)
the
culture.
medium
fetal
calf
CAT
Fig. 1.
was medium
serum
for
18
Cellular
assayed
pCH110)
Eagle's
continued
60
Plasmid
of
(GIBCO
added
10%
a
washed
BRL). or
dexamethasone.
and
on and
modified
further
the pre-
cultured
transfection,
with was
were
and
Dulbecco's
supplemented culture
by
described
confluence
,ƒÊg of
trans-
performed
(GIBCO
OptiMEM,
pCH110
Transient
pMSGCAT
is
promoter,
plasmid
was
until
of
which
MMTV
essentially
OptiMEM
(5 ƒÊg mixed
in
M1.19
(Corning)
cocktail
the
Pharmacia.
procedure [17].
mm
of
expression
obtained
fection
pMSGCAT,
control
and ƒÀ-galactosidase
was
acetyltransferase
assays
and
h
in
extracts
enzyme
the were
activity
as
described [18].
between -188 and —-166,-128 and further downstream of the MMTV probe DNA against DNase I digestion (Fig. 2). The localization of
Results CR-specific vities
of
To
immunoreactivities cytosol
detect
from
the
and
a
rat
DNA-binding
hepatoma
GR-specific
western
immunoblotting
monoclonal
anti-GR
antibodies
the
presence
immunoreactivity molecular
in weight
ponded
to
the
confirmation carried the
out MMTV
1 ƒÊM
a
M1.19
DNase promoter M1.19
dexamethasone,
as cell
the
calculated and
[2,14].
footprint
For GR,
corresfurther we
experiment
probe. cytosol, protected
1
GR-specific
94-kDa
of
the
Figure
single
cytosol, was
presence I
with
a
reports
the
M1.19
we
[14].
of
which
previous of
experiment, with
of
acti-
line
immunoreactivity,
performed
demonstrates
cell
In after
next with
the
present treatment
the
regions
Western immunoblotting of cytosol isolated from a rat hepatoma cell line M 1.19. One-hundred micrograms of cytosol protein were analyzed on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were electrically blotted onto a nitrocellulose filter membrane. Specific interaction between GR protein and anti-rat GR mouse monoclonal antibody was visualized by means of the horse-radish peroxydase-conjugated anti-mouse goat antibody. Molecular weight was determined by the marker run in parallel.
protected regions was shown to be functional with regard to glucocorticoid-inducible transcriptional enhancement (reviewed in [2-4]), and essentially identical to the results from previous experiments in which purified GR were used [15], except for failure to protect the region between -159 and 135. However, similar results to ours were reported by Willmann and Beato with cytosol as a source of GR [10]. Accordingly, these results indicated that cytosol from M1.19 cells contains GR-specific immunoreactivities and glucocorticoid-inducible sequence specific DNA-binding activities.
480
TANAKA
et at.
In vitro activation of cytosol by dexamethasone and monitoring specific DNA-binding activities of GR
concerning GR
the
in
vitro
role
reconstituted
Previous reports have given conflicting results
in
ment
with
from
M1.19
vitro
were
from
these
binding
cytosol
competed
the
indicated
molar
TAT
GRE
encompassing
not
interfere
3B,
see
with
also
legend),
could
be
complex
After gical
concentrations
also
see
incubated on
with
EMSA
incubated
with
probe
urea-polyacrylamide protein G-sequence of I
the
of
gel. cytosol, ladders
downstream
treatment
DNA.
are
M1.19 cytosol preparation)
After
partial
Lane
1,
respectively. run in parallel. footprint. boxed.
(B) Numbers
100 ƒÊg
of
the
using
serum
depict regions
nuleotide
albumin
nucleotide are indicated of
the
MMTV
positions
MMTV
was
DNase
the
treated
I,
absence
samples for
were
control;
positions by a line promoter. relative
lanes
determined except for
the
nM, was
analyzed
amount
hormone in
as
a
the regions
transcription
concen-
(See and
denaturing
50, by
100,
the lower
cyto(lane
probe
on
2-4,
of
and
dexamethasone
analyzed
Protected to
DNA
1 ƒÊm
(5-20
GRE
increased
in
cytosol and
of
an GRE.
physiolo-
vitro,
a trace
promoter with
EMSA
and
nearly
TAT
for did (Fig.
of
probe
the
[20]
our
GR
in
GRE
formation
32P-labeled
that
Although
in
complex
3C)
between
observed
Cytosol by
bovine
sequences
depict
2),
digestion
Numbers Protected DNA
was
SRE
observation
with
for of
sites
or
dexamethasone
TAT
formation
sol
. (A)
the
specific
formation,
cytosol
the this
excess
between
Fig.
(Fig.3C).
complex
with probe
to
be
[19]
for
of
legend
to
indicating
formation of
in
Moreover,
complex
all
unlabeled
binding
relevant
treatment
of present
molar
AP-1
the
(Fig.
between
dose-dependently
shown
factors
transcription
,ƒÊM
DNA-
probe
formed
100-times
the
1 of
GRE
3-6).
oligonucleotides
vitro
experiment Methods" for
a
Cytosol
class
was
was
since
of activi-
32P-labeled
excess
lanes
formation GRE,
cytosol
probe.
single
oligonucleotides 3A,
treat-
of
with
TAT
and
system
I footprint and
the
when
(Fig.
with a
complexes
GRE
TAT
DNase "Materials
a
of
after
presence
incubation
with
amounts
the
we
binding
as
after
activities The
complex
in
of
issue,
GR
EMSA
revealed
reaction
2.
GRE-specific
oligonucleotide
dexamethasone,
Fig.
incubation
the
in
activation this
preparation
and
cells,
the
of
the
monitored
GRE
for address
activation
After
cells
TAT
3A).
ligands To
ligands.
dexamethasone, ties
of
[10-12].
200 ƒÊg
A/G and boundary
from
DNase
start
site.
a
Fig.
3.
Electrophoresis
mobility
was
with
pretreated
cytosol (B)
and
0,
2,
Competition
probe in the oligonucleotides, AP-1;
the
5,
VITRO
ACT1VA
assay
using
32P-labeled
1ƒÊM.1\4 dexamethasone 20,
and
100-fold
experiments. absence
(lane respectively.
at molar
Cytosol
was
10,
[20]. 20
presence
oligonucleotides,
nM
of of
(C)
I,
dexamethasone 20 C1,
nM
probe at
of
protein-DNA
only; 25•Ž
of
pretreated
for
15
dexamethasone complex
tration-dependent fashion when cytosol was treated with increasing concentrations of dexamethasone in vitro (lanes 3-5). To verify the physiological significance of the activation process of GR as represented above, we measured the transcriptional effects of dexamethasone at the corresponding concentrations of dexamethasone using the MMTV-driven CAT reporter plasmid pMSGCAT. After transient transfection of the plasmids, M1.19 cells were treated with the indicated concentrations of de-
30
GR
GRE min.
481
oligonucleotides Lane
unlabeled with
100-times DNA
lanes
OF
TAT for
excess
1) or presence of The non-coding
Lane
HON
25•Ž
5'-CTAGIGATGAGICAGCCGGATC-3'
CTGCGTC-3' 0,
5,
shift
IN
TAT 1ƒÊM
molar sequences
1,
as a probe,
probe
GRE
only;
the AP-1 competitor
incubated 2) or SRE oligonucleotides
SRE;
5'-CTAGAGGATGTCCATATTAGGACAT-
10ƒÊg
of cytosol
and formed;
respectively; 100-fold F,
lane molar
free
was 6,
preincubated 10ƒÊg
excess
of of
of
respectively.
(lane
[19],
Cytosol 10 ƒÊg
then
2-5, min,
(A) 2-6,
oligonucleotides,
dexamethasone,
excess of of these
lanes
in cytosol unlabeled
the
with (lane
presence
the 3) are:
of
preincubated TAT
in GRE
probe.
xamethasone, and the CAT activity of the cellular extracts was determined. Treatment with dexamethasone induced the CAT expression in a concentration dependent fashion (Fig. 4). Together with results from EMSA, we may conclude that, at physiological concentrations of agonistic ligands, GR is activated and allowed to bind its cognate sequences of target genes, and the amounts of activated fraction of GR correlate with the magnitude of subsequent transcriptional effects.
482
Fig.
TANAKA
4.
Expression cells.
of The
galactosidase control for the
lated
cells
concentrations of forms
activity plasmid
were of
rat
hepatoma
pCH110 were
and ƒÀ(as internal transfected by
"Materials in
the
dexamethasone
chloramphenicol
M1.19
pMSGCAT
(see cultured
[14C]-chloramphenicol Of
in
plasmid efficiency)
procedure
and
positions
CAT
expression transfection
lipofectin
Methods") the
the
reporter
and
presence
indicated. (CM) (ACM)
and are
of The acety-
shown.
Discussion We demonstrate here that M1.19 cell cytosol contains cryptic GRE-binding activities that can be dose-dependently induced in, vitro by treatment with dexamethasone. Moreover, these induced DNA-binding activities correlated with in vivo hormonal induction of CAT activity when cells were treated with corresponding concentrations of dexamethasone. In several instances, regulation of the inducible gene expression has been shown to involve posttranslational modification of a pre-existing latent transcription factor to an active form which specifically hinds to regulatory DNA elements in target genes mode
(reviewed in [21]). Here, we show a similar of action of GR which requires ligand-
et al.
dependent conversion in vivo from a non DNAbinding to a DNA-binding form, which we can faithfully mimic in vitro. Although previous studies have dealt with the kinetics between GR and target DNA sequences using purified or expressed fraction as a source of GR [6, 7], it has been very difficult to establish an in vitro activation system for cytosolic GR. One possible explanation for this is that GR protein is labile and susceptible to various in vitro manipulations. Indeed, exposing cytosol to as few in vitro manipulation as possible [13], our system presented here enabled us to monitor activation process of cytosolic GR, since receptor activation is dose-dependently represented by the acquisition of GRE-binding activity. Moreover, we show that the induction of the specific DNAbinding activity in EMSA, reflected the transcriptional potential of the ligands at the corresponding concentrations in vivo. These results therefore favor the idea that receptor activation represents a key regulatory event controlling receptor function. This linear relationship between in vitro activation of cryptic receptor in cytosol and in vivo receptor function strongly argues that hormone effects are likely to be quantitatively regulated at the level of receptor activation and subsequent DNA-binding. Of course, for further confirmation of this, other intracellular events after ligand binding [i.e., nuclear translocation] should also be examined.
Acknowledgments The authors wish to thank Dr. Jan-Ake Gustafsson for generously providing anti-GR antibodies and plasmids. We also thank to Hiromi Oba and Sachiko Iwakami for secretarial assistance. This work was partly supported by the grants to H.T. from Hokkaido Prefecture and the Japan Rheumatism Foundation.
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