(Received for publication, August 1, 1994; and in revised form, October 21, 1994)
From the
The restricted expression of some genes to distinct stages of
the cell cycle is often brought about through alterations in the
activity and/or abundance of specific transcription factors. Many cells
have been shown to be unresponsive to glucocorticoid hormone action
during the G phase of the mammalian cell cycle, suggesting
that some activities of the glucocorticoid receptor (GR), a
ligand-activated transcription factor, are subjected to cell cycle
control. We show here that GR insensitivity in G
is
selective, affecting receptor-mediated transactivation from a simple
glucocorticoid response element, but not repression from a composite
glucocorticoid response element. Since glucocorticoid-dependent
down-regulation of GR protein levels is also unaffected in
G
, distinct activities of the receptor that participate in
this homologous down-regulation must be operating as effectively in
G
-synchronized cells as in asynchronous cells. Finally, the
phosphorylation state of the GR is altered in
G
-synchronized cells reflecting, in part, both
site-specific phosphorylation and dephosphorylation events. These
results suggest that, while GR may be a target for cell cycle regulated
kinases and phosphatases, the resulting changes in receptor
phosphorylation have an impact only on selected GR functions.
Steroid hormones elicit complex responses within cells
predominantly through the action of intracellular receptor proteins
which are members of a large superfamily of nuclear hormone receptors (1) . Members of this superfamily of transcription factors have
the capacity to regulate transcription either via their interactions
with specific target sequences linked to hormonally responsive
genes(2, 3) , or in the absence of direct DNA binding,
by their interactions with other transcription
factors(3, 4) . Given the fairly widespread occurrence
of glucocorticoid receptors (GRs) ()within different tissues
and cell types, multiple cellular factors must influence the
receptor's activity imparting the complexity that often
characterizes physiological responses to glucocorticoids(5) .
In that regard, a number of transcription factors have been identified
that direct cell type-specific (6, 7) and
developmental stage-specific(8, 9) transcriptional
activation activity of the GR.
In many cells which possess the complete machinery necessary to elicit a glucocorticoid response, GR activity can be blunted by the action of other signal transduction pathways. For example, in hepatoma cells glucocorticoid induction of the phosphoenolpyruvate carboxykinase gene can be inhibited by insulin(10, 11) . In addition, activation of protein kinase C by tumor-promoting phorbol esters inhibits glucocorticoid induction of the tyrosine aminotransferase (TAT) gene (12) , illustrating the diversity of intracellular signaling pathways that influence GR action. Since protein kinase C activation in other cases potentiates glucocorticoid induced transcription(13) , additional cell-specific factors must influence the nature of cross-talk between the glucocorticoid and protein kinase C signal transduction pathways. While steroid receptor function has been shown to be influenced by various independent signal transduction pathways which impact upon protein kinases and phosphatases(14, 15, 16, 17) , no detectable alterations in receptor phosphorylation have been observed under these conditions.
Many protein phosphorylation and
dephosphorylation events play critical roles in regulating ordered
progression through the eukaryotic cell
cycle(18, 19) . In some cases, the targets of cell
cycle-regulated kinases and phosphatases have been shown to be
transcription factors whose activity can be altered in numerous ways by
resultant changes in their phosphorylation
state(20, 21, 22) . Tomkins and co-workers
first noted an apparent glucocorticoid insensitivity during the G phase of the cell cycle (23) which has more recently been
shown to reflect, in part, inhibition of GR transactivation
activity(24) . Given the various levels at which gene
expression can be regulated by the
GR(25, 26, 27) , the extent of receptor
activities which may be subjected to cell cycle control remains
unresolved.
We report here our examination of G effects
on GR transactivation and repression brought about through GR
interactions with a promoter-linked simple and composite glucocorticoid
response element (GRE), respectively. Likewise, we have extended our
previous analysis (24) of cell cycle regulation of GR
phosphorylation. Our results reveal that GR insensitivity during the
G
phase of the cell cycle applies to selective GR
functions, i.e. transactivation, but not repression.
Furthermore, we show that complex changes in GR phosphorylation occur
during G
, which include alterations in both overall
phosphorylation and site-specific phosphorylation and
dephosphorylation.
As shown in Fig. 1A, with a cell
synchronization paradigm that uses Hoescht 33342(29) , we were
able to generate a population of GrH2 cells >80% enriched in the
G phase of the cell cycle. This synchronization was readily
reversible (Fig. 1B) as a population of cells 45%
enriched in G
was obtained following a 6-h withdrawal from
medium containing Hoescht 33342. As shown in Fig. 2A,
transcription from a stably integrated TAT3CAT reporter was effectively
induced by dexamethasone in asynchronous GrH2 cells, but not in
G
-synchronized cells. Transcriptional activity of the Rous
sarcoma virus promoter, which is not hormonally
responsive(37) , was not significantly affected by G
synchronization of GrH2 cells (Fig. 2C).
Importantly, the inhibition of GR transactivation from the TAT3CAT
reporter in G
-synchronized cells demonstrates that G
effects on GR transactivation activity apply even when expression
from the GRE-linked promoter is driven by basal components of the
transcriptional machinery.
Figure 1:
FACS analysis of GrH2
cells. Asynchronously growing GrH2 cell cultures (A) were
subjected to G synchrony (B) utilizing Hoechst
33342 as described under ``Materials and Methods.''
Reversibility of the G
arrest is revealed by the FACS
profiles obtained following removal of Hoechst 33342 and culturing
cells for an additional 6 (C) or 10 (D)
h.
Figure 2:
GR
function at a simple, but not composite GRE, is inhibited in
G-synchronized GrH2 cells. Asynchronous (asyn) and
G
-synchronized GrH2 cell lines stably transfected with TAT3 (A), plfG3 (B), or Rous sarcoma virus long terminal
repeat (C) CAT reporter plasmid were either untreated(-)
or treated (+) with 0.1 µM dexamethasone (dex) for 6 h. CAT assays were performed using equivalent
amounts of protein in crude cell-free lysates. Average CAT activity (n = 3) is expressed relative to that obtained in
untreated asynchronous cells. The 30 and 50% dexamethasone repression
of plfG3 CAT activity observed in asynchronous and
G
-synchronized cells, respectively, was statistically
significant (p < 0.005) as determined using a two-tailed
Student's t test.
GRs are bifunctional transcription
factors which can act, in certain contexts, as transcriptional
repressors as well as
activators(36, 38, 39, 40) . While
various mechanisms account for GR-mediated transcriptional
repression(4) , a composite GRE (cGRE) located within the rat
proliferin gene can direct the GR to activate or repress transcription
from a linked promoter depending upon the composition of AP-1
transcription factors (41) that co-occupy the
cGRE(36) . To compare the activity of the proliferin cGRE with
a simple GRE in G-synchronized cells, GrH2 cells were
stably transfected with a reporter (plfG3) (36) possessing the
proliferin cGRE linked to the identical Drosophila alcohol
dehydrogenase minimal promoter that was used to monitor the activity of
a simple GRE. As shown in Fig. 2B, plfG3 promoter
activity was repressed upon dexamethasone treatment of both
asynchronous and G
-synchronized GrH2 cells. The extent of
dexamethasone repression of plfG3 promoter activity was similar in
asynchronous and G
-synchronized GrH2 cells, despite the
fact that basal activity of the plfG3 promoter was reduced in
G
-synchronized cells (Fig. 2B). Thus GRs
are not completely disabled in G
-synchronized cells, and
although they are severely compromised in their ability to activate
transcription, they still retain the capacity to repress transcription.
Figure 3:
GR protein levels are similarly
down-regulated in response to dexamethasone (dex) treatment of
asynchronous and G-synchronized GrH2 cells. Asynchronous
and G
-synchronized GrH2 cells were treated with 0.1
µM dex for the lengths of time indicated (in hours). GRs
were immunoprecipitated from the same amount of total protein in whole
cell extracts, electrophoresed on 7.5% polyacrylamide-SDS gels, and
transferred to nitrocellulose. GR protein was visualized as described
under ``Materials and Methods.'' The migration of protein
molecular mass standards (in kilodaltons) is indicated. The intensely
stained rapidly migrating band represents the heavy chain of the BuGR2
antibody which was used to immunoprecipitate the GR and is detected
upon development of the Western blot.
Glucocorticoid-induced hyperphosphorylation of the GR has been
observed in mouse (44, 45) and rat cells (30) and reflects increased overall phosphorylation (Table 1) (44) as well as hyperphosphorylation at
specific sites(30) . As seen in Fig. 4(compare Panels A and B), dexamethasone treatment of GrH2
cells led to prominent hyperphosphorylation of a few specific peptides
(peptides g and h), in agreement with our observations in other
cultured cell lines(14, 15, 30) , and
prominent dephosphorylation of another peptide (peptide c). Other minor
variations in spot intensity were not reproducible in GrH2 cells or in
different cell lines that we have previously
examined(14, 15, 30) . Despite the fact that
overall GR phosphorylation was not increased upon dexamethasone
treatment of G-synchronized cells (Table 1), the
identical prominent hyperphosphorylation of GR peptides g and h was
observed in G
-synchronized GrH2 cells (Fig. 4,
compare Panels C and D). In fact, the only striking
difference in GR phosphorylation between asynchronous and
G
-synchronized cells was observed on peptide d. In the
absence of dexamethasone treatment, peptide d was hyperphosphorylated
2-fold in G
-synchronized cells relative to asynchronous
cells (Fig. 4, compare Panels A and C). The
closely migrating peptide c was shown to be phosphorylated to the same
extent in asynchronous and G
-synchronized cells using
identical means for quantifying phosphorylation within individual
peptides (see ``Materials and Methods''). It seems unlikely
that this hyperphosphorylation accounts entirely for increased overall
phosphorylation of GR observed in G
-synchronized cells (Table 1), as we have previously determined that this peptide
accounts for only approximately 10% of total GR phosphorylation in rat
cell lines (not shown). In addition to this change in constitutive
phosphorylation of peptide d, dexamethasone treatment of
G
-synchronized cells led to a dephosphorylation of this
peptide (Fig. 4, compare Panels C to D). Using
a peptide whose level of phosphorylation was not significantly affected
by dexamethasone as a reference, the relative extent of peptide d
phosphorylation was shown to be reduced by 60% upon dexamethasone
treatment of G
-synchronized cells. A less dramatic
dephosphorylation of peptide d was noted upon dexamethasone treatment
of asynchronous cells (Fig. 4, compare Panels A to B). Thus, the lack of overall hormone-induced
hyperphosphorylation of GRs in G
-synchronized cells is
misleading as analysis of GR phosphorylation at specific sites revealed
both hormone-induced phosphorylation and dephosphorylation during
G
.
Figure 4:
Site specific alterations in GR
phosphorylation and dephosphorylation in G-synchronized
GrH2 cells.
P-Labeled GR protein was isolated from
asynchronous and G
-synchronized GrH2 cells treated or
untreated with 0.1 µM dexamethasone for 1 h and subjected
to two-dimensional tryptic mapping on thin layer cellulose plates. A, B, C, and D show autoradiographs
of corresponding tryptic maps from untreated asynchronous (A),
dexamethasone-treated asynchronous (B), untreated
G
-synchronized (C), and dexamethasone-treated
G
-synchronized (D) samples. Phosphopeptides g and
h (A) become hyperphosphorylated upon dexamethasone treatment
of asynchronous (B) and G
-synchronized (D) cells (highlighted by filled horizontal arrows in B and D). Phosphopeptide c (A) is relatively
dephosphorylated upon dexamethasone treatment of asynchronous (B) and G
-synchronized (D) cells (unfilled, vertical arrows in B and D). Phosphopeptide d (A) is hyperphosphorylated in
G
-synchronized cells relative to asynchronous cells (filled, vertical arrowhead in C) but
becomes dephosphorylated upon dexamethasone treatment of
G
-synchronized cells (unfilled, vertical
arrowhead in D). Analogous results were obtained in
another independent tryptic mapping
experiment.
There are a limited number of genes whose expression is
restricted to specific stages of the cell cycle(46) . This
feature applies both to genes whose products serve an important
regulatory role in cell cycle progression, such as G(47) and G
(48) cyclins, and to
others which encode proteins more intimately involved in the mechanics
of the chromosomal replication (49) condensation(50) ,
and segregation(51) . Alterations in the abundance and/or
activity of the transcription factors that are required for efficient
expression of cell cycle regulated genes often accounts for their
restricted expression(46) . Our previous work(24) , and
that reported herein, establish that GR transactivation activity is
severely impaired in G
-synchronized cells, even from a
promoter that utilizes only basal components of transcription
machinery. It therefore seems unlikely that G
-specific
alterations in the activity of transcription factors required for
efficient glucocorticoid induction (35) are solely responsible
for the refractoriness of endogenous promoters to GR transactivation.
Since GR in crude nuclear extracts prepared from
G
-synchronized GrH2 cells can bind DNA efficiently in
vitro, (
)we likewise do not believe that the failure to
detect glucocorticoid induction is due to inhibition of GR binding to
target GREs.
A novel finding presented in this report is that not
all nuclear functions of GR are disrupted as GR-mediated repression
directed by a cGRE is apparently unaffected in
G-synchronized cells. The mechanism of repression in this
case involves the co-occupancy at the cGRE of GR with a specific cohort
of AP-1 family members(36) , further supporting the notion that
GR DNA binding activity is not grossly affected by G
synchronization. The expression of this transcriptional
modulatory function of the GR argues against the possibility that
receptors are sequestered during G
within a subnuclear
compartment (52) that restricts their ability to productively
interact with active transcription factors (i.e. AP-1), or
that chromatin structural changes in G
render all target
sites inaccessible to the GR. Since the proliferin cGRE can also direct
GR to activate transcription from linked promoters (36) in the
appropriate context, it would be interesting to examine whether GR
transactivation from that element is likely impaired during
G
.
In G-synchronized cells, quantitative
effects on GR phosphorylation and dephosphorylation were observed.
Hormone treatment of both asynchronous and G
-synchronized
cells led to dephosphorylation of a single peptide (peptide c), while
more extensive dephosphorylation of a different peptide (peptide d) was
observed in hormone treated G
-synchronized cells. Since
peptide d is hyperphosphorylated in G
-synchronized cells
not treated with hormone, the phosphorylation state of GR in hormone
treated G
cells appears remarkably similar to that obtained
from asynchronous cells. Thus, basal phosphorylation of the GR, and not
its phosphorylation state in hormone treated cells, is what
distinguishes the receptor in asynchronous versus G
-synchronized GrH2 cells. If increased basal
phosphorylation of GR is responsible for the inhibition of its
transactivation activity, perhaps the GR, analogous to other
transcription factors such as c-jun(53) , possesses
sites that can exert an inhibitory effect on its activity when
phosphorylated. The inability of hormone-induced dephosphorylation of
GR in G
to overcome this inhibition could be explained if
this dephosphorylation occurs subsequent to receptor interactions with
the transcriptional machinery.
The amino acid sequences surrounding
most mouse GR phosphorylation sites (54) implies that they are
likely to be the targets of proline-directed protein kinases which
could include members of both the cyclin-directed and mitogen-activated
protein (MAP) kinase families (55) . Obviously, any members of
this family that are activated during G(56) could
account for the site-specific hyperphosphorylation of GR in cells not
treated with hormone. Regardless of the identity such GR kinase(s), the
resultant hyperphosphorylation of GR does not affect all its nuclear
functions, and appears to be associated only with inhibition of
transactivation. Both PP-1 and PP-2A have been shown to dephosphorylate
specific GR sites in vitro and are implicated in the in
vivo dephosphorylation of GR(30) . In fact, the
hormone-induced dephosphorylation of GR occurs at sites that are major in vitro substrates for PP-1 and PP-2A. Although we have not
detected differences in PP-1 and PP-2A activity in crude cell extracts
prepared from asynchronous and G
-synchronized cells,
specific phosphatase targeting subunits (57) could be
involved in regulating their activity on GR during G
. Since
hormone-induced dephosphorylation in G
restores a normal GR
phosphorylation pattern but not transactivation, protein phosphatases
may act at a step in the nuclear processing of GR that follows receptor
interactions with the transcriptional machinery. This notion is
supported by the fact that a population of GR that is relatively
dephosphorylated has been found to be confined to a distinct subnuclear
compartment, defined biochemically(58) .
The diversity of
endogenous and synthetic glucocorticoid-responsive genes whose
induction is inhibited in G(23, 24, 59) (this report) implicates
the receptor as a likely target of G
-specific regulators.
While GR interactions with components of the transcriptional machinery
that participate in transactivation may be sensitive to some
G
-specific biochemical alteration in the receptor,
interactions of the receptor with distinct transcription factors that
bring about transcriptional repression must be transparent to these
biochemical changes. Our results point out the potential influence of
both kinases and phosphatases in regulating GR activity, and by
demonstrating the specificity in affected GR functions, illustrate the
diversity of effects that intracellular signaling pathways could exert
on steroid hormone responsiveness.