Glucocorticoid Inhibition of Fibroblast Proliferation and Regulation of the Cyclin Kinase Inhibitor p21Cip1
Arivudainambi Ramalingam,
Aki Hirai and
E. Aubrey Thompson
Department of Human Biological Chemistry and Genetics
University of Texas Medical Branch Galveston, Texas 77550-0645
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ABSTRACT
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Glucocorticoids inhibit the proliferation of
fibroblastic cells in vivo and in culture; however, the
molecular mechanism that accounts for this effect has remained obscure.
We have undertaken to elucidate the mechanism whereby glucocorticoids
decrease the rate of proliferation of mouse L929 fibroblastic cells.
Addition of dexamethasone to mid-log phase fibroblasts prolongs G1
phase. This increase in the G1 interval is associated with, and
probably due to, inhibition of phosphorylation of the product of the
Rb-1 tumor suppressor gene, pRb. Inhibition of pRb
phosphorylation by cyclin D-dependent kinases can be demonstrated
in vitro. Nevertheless, there is no detectable change in
the expression of cyclin D1, cyclin D2, or cyclin D3. Cyclin-dependent
kinase-4 (Cdk4) and Cdk6 are not down-regulated in L929 cells after
addition of glucocorticoids, and the abundance of cyclin D/Cdk4
complexes does not change. Inhibition of pRb kinase activity is
associated with an increase in the abundance of one of the Cdk
inhibitors, p21Cip1. The abundance of another
cyclin kinase inhibitor, p27Kip1, remains
constant. The amount of Cdk4 that is bound to
p21Cip1 increases rapidly after addition of
dexamethasone, and the activity of Cdk4-pRb kinase decreases in
parallel. These results indicate that glucocorticoid inhibition of
fibroblast proliferation is due to induction of
p21Cip1, which binds to and inactivates
cyclinD/Cdk4 complexes. The abundance of p21 mRNA increases about
5-fold within 2 h after addition of dexamethasone. This effect
does not obtain in L929 mutants that are null for the glucocorticoid
receptor, and a variant that expresses the glucocorticoid receptor from
a tetracycline-repressible expression vector demonstrates induction of
p21 mRNA only in the absence of tetracycline. Cycloheximide does not
block induction of p21 mRNA, and dexamethasone has no detectable effect
on the apparent rate of degradation of p21 mRNA. Nuclear run-on
transcription of the Cip1 gene increases within 2 h
after addition of dexamethasone. This effect can be blocked by
tetracycline-mediated repression of the glucocorticoid receptor.
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INTRODUCTION
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Regulation of fibroblast proliferation is critical for
regeneration of wounded tissue. Fibroblast proliferation at wounded
sites, as well as other sites in the body, is stimulated by a large
number of hormones, including platelet-derived growth factor,
fibroblast growth factors, and transforming growth factors.
Glucocorticoids, on the other hand, inhibit fibroblast proliferation
and delay wound healing. The mechanism of glucocorticoid inhibition of
fibroblast proliferation is unclear, and the data on the effects of
glucocorticoids on fibroblast proliferation in culture are sometimes
conflicting (reviewed in Ref.1). In general, low concentrations of
glucocorticoids potentiate the mitotic stimulation that occurs when
growth factors are added to serum-starved fibroblasts. On the other
hand, higher concentrations of glucocorticoids inhibit proliferation of
mid-log phase cultures of fibroblasts.
Naturally occurring and synthetic glucocorticoids inhibit proliferation
of mouse L929 fibroblasts in culture (2, 3, 4, 5). Addition of
10-7 M dexamethasone to mid-log L929 culture
causes an accumulation of cells with a G1 DNA content (5). Similar
results have been observed in glucocorticoid-treated lymphoid cells
(6). Inhibition of G1 progression in lymphoid cells is due to the
effects of glucocorticoids upon G1 cyclin gene expression (7, 8, 9), and
we have undertaken to test the hypothesis that a similar mechanism
prevails in fibroblasts.
Progression through the G1 phase of the cell cycle is regulated by a
family of serine/threonine protein kinases called cyclin-dependent
kinases or Cdks, which are activated by association with a stimulatory
subunit, a cyclin (reviewed in Refs. 1013). Two families or classes
of cyclins interact with at least three different Cdks during G1
progression: members of the D cyclin family (cyclin D1, cyclin D2, and
cyclin D3) bind to and activate Cdk4 and Cdk6, whereas cyclin E
activates Cdk2.
Inhibition of Cdk activity is mediated, in part, by a second set of
regulatory subunits, the cyclin kinase inhibitors (reviewed in Refs.
1416) There are two distinct classes of cyclin kinase inhibitors.
Relatives of INK4 bind to Cdk4- and Cdk6-containing complexes, causing
dissociation of the cyclin and inhibition of kinase activity (17, 18).
One of these inhibitors, p15INK4B, is thought to play a
role in TGFß inhibition of keratinocyte proliferation (19, 20). A
second family of cyclin kinase inhibitors is comprised of relatives of
p21Cip1. Members of this family bind to Cdk2-, Cdk4-, and
Cdk6-containing complexes, prevent their activation at low
stoichiometry, and block kinase activity at high stoichiometry
(21, 22, 23, 24). Induction of p21Cip1 is involved in p53-mediated
cell cycle arrest (25), in proliferative senescence (26), in
replication and repair of damaged DNA (27, 28), and during
differentiation of specific tissue types (29, 30, 31). A relative of
p21Cip1 called p27Kip1 is thought to be
involved in contact inhibition (32), cAMP inhibition of cell
proliferation (33), and TGFß inhibition of epithelial cell
proliferation (20, 32, 34, 35).
It is believed that cyclin kinase inhibitors block cell proliferation
by virtue of their ability to inhibit Cdk-dependent phosphorylation of
critical substrates in the G1 phase of the cell cycle (reviewed in
Refs. 1416). Both Cdk2 and Cdk4 can phosphorylate tumor suppressor
gene products, including the product of the Rb-1 gene, pRb,
and the related protein p107 (3639). In cells that express wild type
pRb, phosphorylation of pRb is necessary for initiation of S phase
(40, 41, 42), and cells that harbor Rb-1 mutations do not
require cyclin D for G1 progression (43, 44, 45, 46). The preponderance of
evidence indicates that phosphorylation of tumor suppressor gene
products by cyclin D/Cdk4 may be central to the role of these kinases
in regulating cell cycle progression. Cyclin E/Cdk2 kinase probably
plays a role in activating E2F-like transcription factors which, in
turn, control nucleotide metabolism in late G1 and S phase (reviewed in
Refs. 47 and 48).
The D type cyclins, in conjunction with Cdk4 or Cdk6, appear to be
primary targets for hormones that stimulate cell proliferation. D type
cyclins are induced by a large number of growth factors (49, 50, 51), and
abrogation of D cyclin induction blocks the mitogenic effects of such
hormones (52, 53). Hormones that inhibit cell proliferation, such as
TGFß, cause a decrease in the amount or activity of cyclin D/Cdk4
complexes (reviewed in Ref.54). Glucocorticoid inhibition of lymphoid
cell proliferation is due, in part, to inhibition of cyclin D3
expression (7, 8). We speculated that a similar mechanism might account
for glucocorticoid inhibition of fibroblast proliferation.
The experiments described below were undertaken to test the hypothesis
that glucocorticoids cause a decrease in the amount or activity of
cyclin D/Cdk4 kinase complexes in fibroblasts. We have assayed pRb
phosphorylation in vivo and in vitro. pRb kinase
activity has been correlated with expression of the catalytic and
regulatory subunits of cyclin D1- and D3-dependent kinases. In
addition, we have assayed for the expression of two well characterized
cyclin kinase inhibitors, p21Cip1 and p27Kip1.
The data indicate that glucocorticoids regulate cyclin D/Cdk4 kinase
activity in fibroblasts. However, the mechanism is fundamentally
different from that described for glucocorticoid inhibition of lymphoid
cell proliferation.
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RESULTS
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Glucocorticoid Inhibition of pRb Phosphorylation
Hyperphosphorylation of pRb is associated with, and may be
necessary for, G1 progression and initiation of S phase (40, 41, 42). A
large number of phosphate residues are attached to pRb, resulting in a
significant degree of electrophoretic polymorphism (40, 41, 42).
Consequently, the extent of phosphorylation can be assayed by Western
blotting. As shown in Fig. 1
, pRb from mid-log phase L929
cells resolved as a number of species with different electrophoretic
mobilities. pRb from dexamethasone-treated cells was much less
polymorphic and tended to migrate more rapidly than some of the pRb
species that were detected in extracts from untreated mid-log phase
cells. These properties are consistent with the conclusion that the
state of pRb phosphorylation is reduced in glucocorticoid-treated L929
cells.

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Figure 1. pRb Phosphorylation in Vivo in L929
Cells
Mid-log phase L929 cells were treated with ethanol (control) or
dexamethasone (10-7 M). Cell extracts were
collected after 24 h and analyzed for the state of pRb
phosphorylation by Western blotting.
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Glucocorticoid inhibition of pRb kinase activity could be demonstrated
in vitro. Extracts from control and dexamethasone-treated
L929 cells were immunoprecipitated with antibodies against cyclin D1
and cyclin D3 so as to precipitate cyclin D/Cdk4 complexes. These
complexes were then assayed for their ability to phosphorylate
recombinant glutathione-S-transferase-Rb(GST-Rb) in
vitro, as shown in Fig. 2
. Both cyclin D1- and cyclin
D3-associated pRb kinase activity was inhibited by more than 80% after
addition of dexamethasone to mid-log phase L929 cells.

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Figure 2. Cyclin D1- and Cyclin D3-Associated pRb Kinase
Activity in Vitro
Control and dexamethasone-treated L929 cell extracts were
immunoprecipitated with antibodies against cyclin D1 or D3 so as to
precipitate the cyclin/Cdk complexes. These complexes were assayed for
their ability to phosphorylate recombinant GST-Rb in
vitro. The reaction mixture was resolved on polyacrylamide gel
and exposed to x-ray film.
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Glucocorticoids Effects on Expression of G1 Cyclins and Cdks
Phosphorylation of pRb is catalyzed during early to mid-G1 by a
complex consisting of one of the D-type cyclins and either Cdk4 or
Cdk6. Glucocorticoids inhibit D3 cyclin and Cdk4 expression in lymphoid
cells (7, 9), and a corresponding decrease in pRb kinase is observed in
dexamethasone-treated lymphoid cells (8). Such observations suggest
that glucocorticoid inhibition of cyclin D1- and cyclin D3-associated
pRb kinase in L929 cells could be due to down-regulation of the
regulatory or the catalytic subunit of the G1 pRb kinase
(i.e. cyclin D or Cdk4). To test this hypothesis, D cyclins
and G1 Cdks were assayed in extracts from control and
glucocorticoid-treated L929 cells. As shown in Fig. 3A
, dexamethasone had no effect on the abundance of Cdk4, Cdk6, cyclin D1,
cyclin D2, or cyclin D3. These data are inconsistent with the
hypothesis that prolongation of G1 phase in glucocorticoid-treated
cells is due to inhibition of D cyclin or Cdk expression. In this
respect fibroblasts are very different from lymphoid cells (7, 8, 9).

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Figure 3. Abundance of G1 Cyclins and Cdks and Cyclin/Cdk
Complexes in Glucocorticoid-Treated Cells
In the experiment shown in panel A, control and
dexa-methasone-treated L929.06 cells were harvested 24 h after
treatment. The extracts were assayed for abundance of cyclin D1, cyclin
D2, cyclin D3, Cdk4, and Cdk6 by Western blotting. Panel B, Mid-log
L929.06 cells were treated with dexamethasone, and extracts were
prepared 0, 6, and 24 h after treatment. Cell extracts were
immunoprecipitated with antibodies against cyclin D1 or cyclin D3.
Immunoprecipitated complexes were dissolved, resolved by
electrophoresis, and assayed for Cdk4 by Western blotting.
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Although glucocorticoids do not affect the abundance of G1
cyclins or Cdks, they might directly or indirectly affect the ability
of the D cyclins to bind to their respective Cdks and thereby inhibit
the formation of active cyclin D-dependent kinase complexes. To test
this hypothesis, control and glucocorticoid-treated L929 cell extracts
were immunoprecipitated with antibodies against cyclin D1 and D3. The
immunoprecipitates were used to assay Cdk4 to quantify the amount of
cyclin D/Cdk complexes in glucocorticoid-treated cells. As shown in
Fig. 3B
, there was no decrease in the amount of Cdk4 that could be
coimmunoprecipitated with cyclin D1 or D3. This observation indicates
that glucocorticoids do not inhibit the association of D cyclins with
Cdk4 in L929 cells.
Glucocorticoid Effects on Cyclin Kinase Inhibitors
p21Cip1 and p27Kip1
Cyclin D1- and D3-associated pRb kinase activity is
decreased with no corresponding decrease in the abundance of either
catalytic or regulatory subunits in glucocorticoid-treated L929 cells.
This observation suggests that glucocorticoids may cause an increase in
the amount or activity of cyclin kinase inhibitors, such as
p21Cip1 or p27Kip1. The abundance of these two
inhibitors was measured in control and dexamethasone-treated L929
cells. As shown in Fig. 4
, the abundance of p27 was
relatively high in mid-log phase L929 fibroblasts, and addition of
glucocorticoids had no significant effect upon p27 abundance. Unlike
p27, the abundance of p21 was very low in mid-log phase L929 cells
(Fig. 4
). Furthermore, dexamethasone induced p21 expression. The
abundance of this inhibitor increased by 5- to 10-fold within 6 h
and remained high as long as cells were exposed to the steroid.

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Figure 4. Abundance of Cyclin Kinase Inhibitors p21 and p27
in Glucocorticoid-Treated Cells
Mid-log L929.06 cells were treated with dexamethasone. Extracts were
collected 0, 6, and 24 h and analyzed for cyclin kinase inhibitors
p21 and p27 by Western blot analysis.
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p21Cip1 Binding to Cdk4 Complexes and
Effects on Rb Kinase Activity
The observed increase in the abundance of p21 in
glucocorticoid-treated L929 cells suggests the possibility that this
kinase inhibitor might bind to G1 cyclin/Cdk complexes and block the
kinase activity in L929 fibroblasts. To test whether this increase in
the abundance of p21 resulted in an increase in the abundance of
p21/Cdk4 complexes, control and glucocorticoid-treated L929 cell
extracts were immunoprecipitated with a polyclonal antibody against
p21. The abundance of Cdk4 in these immunoprecipitates was assayed by
Western blotting. Binding of p21 to Cdk4 increased rapidly, as p21 was
induced in glucocorticoid-treated cells. As shown in Fig. 5A
, the abundance of p21/Cdk4 complexes increased 5- to
10-fold within 6 h and remained constant thereafter.

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Figure 5. Effect of Glucocorticoids on Cdk4-Associated p21
and Rb Kinase Activity
In the experiment shown in panel A, extracts were prepared from cells
treated with dexamethasone for 0, 6, and 24 h. Proteins were
immunoprecipitated with antibody against p21. The immunoprecipitated
complexes were analyzed for the presence of Cdk4 by Western blotting.
Panel B, Extracts used in the experiment shown in panel A
immunoprecipitated with antibody against Cdk4. The complexes were then
assayed for their ability to phosphorylate recombinant GST-Rb in
vitro.
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In parallel experiments, the same extracts that were used in the
experiment shown in Fig. 5A
were immunoprecipitated with a polyclonal
antibody against Cdk4. These immunoprecipitates were assayed for their
ability to phosphorylate GST-Rb in vitro. As shown in Fig. 5B
, the kinase activity associated with Cdk4 decreased significantly
within 6 h after addition of dexamethasone and remained low
thereafter.
Glucocorticoid Induction of p21Cip1
mRNA
In an effort to understand the mechanism whereby glucocorticoids
induce p21 expression, RNA was isolated from dexamethasone-treated L929
cells, and the abundance of p21 mRNA was ascertained as shown in Fig. 6A
. The abundance of p21 mRNA increased 4- to 6-fold within
2 h after addition of dexamethasone. No such induction was
observed when dexamethasone was added to E82.A3 cells (Fig. 6B
), a
variant of L929 that is null for the glucocorticoid receptor (55). The
expression of p21 mRNA was analyzed in a derivative of E82.A3 that was
engineered to expresses the mouse glucocorticoid receptor from a stably
integrated, tetracycline-repressible expression vector. This cell line
is called E82T4/GR3. As shown in Fig. 6C
, p21 mRNA was rapidly induced
when dexamethasone was added to E82T4/GR3 cells that had been
maintained in the absence of tetracycline, so as to de-repress
expression of the glucocorticoid receptor. However, no such induction
obtained when dexamethasone was added to cells that had been maintained
in the presence of 0.1 µM tetracycline for 3 days before
addition of the steroid.

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Figure 6. Glucocorticoid Induction of p21 mRNA
In the experiment shown in panel A, mid-log phase L929.06 cultures were
treated with dexamethasone, and mRNA was extracted at the intervals
indicated. Twenty micrograms of total RNA from each sample were
resolved by electrophoresis and quantified by hybridization to labeled
probes corresponding to mouse Cip1 cDNA or mouse 18S rRNA. A similar
experiment was repeated in panel B using E82.A3 cells, which are null
for the glucocorticoid receptor. E82T4/GR3 cells were analyzed in the
experiment shown in panel C. Parallel cultures were maintained in the
presence or absence of 0.1 µg/ml tetracycline. The cells were treated
with dexamethasone and analyzed for p21 mRNA abundance, as shown.
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The abundance of p21 mRNA reproducibly decreased to near basal
expression within 24 h after addition of dexamethasone to wild
type L929 cells, as shown in Fig. 6A
. On the other hand, glucocorticoid
induction of p21 mRNA was more robust and more persistent in E82T4/GR3
cells, when such cells were exposed to dexamethasone in the absence of
tetracycline (Fig. 6C
). Glucocorticoid-mediated down-regulation of the
glucocorticoid receptor is characteristic of L929 cells (56) and might
account for the transitory pattern of p21 mRNA induction. Figure 7
shows Western blotting data in which the glucocorticoid
receptor was measured in those L929 variants that were analyzed for p21
induction. The first four lanes contain the results from the
tetracycline-repressible E82T4/GR3 cells, and lanes 1 and 3 reveal the
extent to which the glucocorticoid receptor is de-repressed by
withdrawal of tetracycline. The abundance of the receptor in the fully
de-repressed E82T4/GR3 cells (lane 3) was at least 5 times greater than
that observed in wild type L929 cells (lane 5). The expression of the
glucocorticoid receptor decreased after addition of dexamethasone to
both wild type (lane 6) and E82T4/GR3 cells (lane 4), but the abundance
of the receptor in the E82T4/GR3 cells 24 h after dexamethasone
(lane 4) was equal to or greater than that observed in control L929
cells (lane 5). This apparent overexpression of the glucocorticoid
receptor may account for the extent to which the induction of p21 mRNA
persists after addition of dexamethasone to E82T4/GR3 cells (as shown
in Fig. 6C
).

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Figure 7. Regulation of Glucocorticoid Receptor Expression in
L Cell Lines
Extracts were prepared from mid-log phase cells or from cultures that
had been treated for 24 h with dexamethasone. In addition,
cultures were prepared from E82T4/GR3 cells that had been maintained in
the presence of 0.1 µg/ml tetracycline (lanes 1 and 2). Forty
micrograms of total cellular protein were resolved by electrophoresis,
blotted to nitrocellulose filters, and probed with BUGR
anti-glucocorticoid receptor antibody, as described in Materials
and Methods. The predicted position of the 94-kDa
glucocorticoid receptor is indicated by the arrow.
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Induction of Cip1 Transcription in
Glucocorticoid-Treated Cells
The data shown in Figs. 6
and 7
indicate that induction of p21
expression in L929 cells depends upon expression of the glucocorticoid
receptor. Furthermore, induction of p21 mRNA was not blocked when cells
were exposed to cycloheximide for 2 h before addition of
dexamethasone, as shown in Fig. 8A
. Neither did
dexamethasone alter the apparent rate of degradation of p21 mRNA, as
shown in Fig. 8B
. In this experiment, the abundance of p21 mRNA was
measured at intervals after addition of actinomycin D to control L929
cells and to cells that had been treated with dexamethasone for 2
h before addition of actinomycin D. The apparent half-life of p21 mRNA
was 46 h, irrespective of the presence of glucocorticoids. The
observation that p21mRNA stability is not altered by dexamethasone
suggests that glucocorticoids must increase transcription of
Cip1; moreover, the observation that induction of p21 mRNA
cannot be blocked by cycloheximide suggests that such induction may be
due to a direct interaction between the Cip1 promoter and
the glucocorticoid receptor. Nuclear run-on transcription was carried
out to determine whether induction of p21 mRNA was associated with
increased Cip1 transcription, as shown in Fig. 8C
.
Cip1 transcription was increased after addition of
dexamethasone to E82T4/GR3 cells. When the data from three independent
experiments were quantified and normalized to transcription of the 5S
RNA gene (as shown in the bar graph in Fig. 8C
), the mean
stimulation was estimated to be 4- to 6-fold, consistent with the
increase in p21 mRNA that one observes within 2 h after addition
of dexamethasone. The glucocorticoid-mediated increase in
Cip1 transcription was blocked when cells were grown in the
presence of tetracycline, so as to repress the glucocorticoid receptor,
before addition of dexamethasone (Fig. 8C
).

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Figure 8. Effects of Cycloheximide and Actinomycin D on p21
mRNA Expression
In the experiment shown in panel A, mid-log phase cultures were treated
for 2 h with 5 µg/ml cycloheximide, as indicated. Dexamethasone
was added and RNA was extracted after 2 h in the presence of
steroid. The abundance of p21 mRNA was assessed by Northern blotting,
using 20 µg total cellular RNA. In the experiment shown in panel B,
parallel cultures were prepared. One was treated for 2 h with
dexamethasone () while the other was untreated ( ). Actinomycin D
was added to a final concentration of 1 µg/ml, and RNA was extracted
at intervals thereafter. The abundance of p21 mRNA was determined by
Northern blotting, and the data were quantified by densitometry. Curves
depicting decay kinetics were fitted by first-order linear regression
analysis.
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DISCUSSION
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The product of the retinoblastoma susceptibility gene, pRb, is the
prototypic tumor suppressor protein. Although the mechanism of action
of pRb is far from understood, there are certain general features of
this regulatory entity that may be perceived. It would appear that pRb
blocks progression through the G1 phase of the cell cycle. This
blockade is relieved by extensive phosphorylation, which occurs during
mid- to late G1 phase (3742, 5759). Available data are consistent
with the hypothesis that phosphorylation of pRb releases an array of
proteins including, but probably not limited to, transcription factors
such as E2F-1 and UBF-1 (47, 60). These factors then stimulate or
sustain an ordered reaction pathway that culminates in the cellular
commitment to initiate DNA replication. When cells fail to
phosphorylate pRb, they will arrest in G0/G1 phase (48).
Several enzymes phosphorylate pRb. Cdk4 and Cdk6 phosphorylate pRb and
the related pocket protein p107 (61, 62), and there are data that
suggest that these enzymes have no other essential function (43, 44, 45, 46).
Cdk2 can also phosphorylate pRb (63, 64, 65, 66), but there is controversy
concerning the extent to which Cdk2-dependent phosphorylation of pRb is
essential to cell cycle progression (67). Despite an incomplete
understanding of the enzymology, it is clear that hormonal regulation
of cell proliferation impinges upon pRb phosphorylation. A wide variety
of growth-promoting hormones regulate the expression of one or another
of the cyclin subunits that are required for activation of the pRb
kinases. This type of regulation was first shown for CSF-1, which is
necessary for expression of cyclin D1 in myelogenous cells (49).
Virtually every growth-promoting hormone that has been examined to date
stimulates the expression of one of the D-type cyclins (61, 68). The
data are consistent with the proposition that members of the D cyclin
family are among the primary targets for all mitogenic hormones.
Phosphorylation of pRb is also regulated by hormones that inhibit cell
proliferation. TGFß inhibits pRb phosphorylation and causes G1 arrest
in sensitive epithelial cells (69), whereas glucocorticoids inhibit pRb
phosphorylation and cause G1 arrest in murine lymphoma cells (8). Both
hormones cause a decrease in the amount or the activity of a cyclin
D/Cdk4 (or cyclin D/Cdk6) pRb kinase, although the effects of TGFß
depend to an extent upon the cell line that is studied (19, 20, 54, 70). The mechanism of inhibition of pRb phosphorylation is fairly
straightforward in glucocorticoid-arrested T lymphoma cells.
Glucocorticoids inhibit the expression of the major D-type cyclin in
cells of thymic origin, i.e. cyclin D3 (7). The activity of
Cdk4 (and probably Cdk6) decreases in consequence and the cells arrest
in G1 (9). Antiestrogens inhibit expression of cyclin D1 in breast
cancer cells (71), and there are data that suggest that glucocorticoid
inhibition of Con8 breast cancer cell proliferation may involve changes
in cyclin D1 expression (72). Taken together, these data suggest that
steroid hormones that inhibit cell proliferation may do so by virtue of
their ability to down-regulate one of the members of the D cyclin
family.
Glucocorticoid inhibition of fibroblast proliferation appears to
involve a different mechanistic strategy. Glucocorticoids inhibit pRb
phosphorylation, and everything that we know about cell cycle
progression indicates that this effect is sufficient to block entry
into S phase. Moreover, inhibition of pRb phosphorylation is due to a
glucocorticoid-mediated decrease in the activity of cyclin D/Cdk4.
Thus, glucocorticoids regulate the proliferation of both lymphoid and
fibroblastic cells by a common device: inhibition of cyclin D/Cdk4 pRb
kinase activity. Unlike the situation that obtains in lymphoid cells,
one does not observe any significant change in the expression of any of
the D cyclins or their catalytic partners, Cdk4 or Cdk6, in
glucocorticoid-treated L929 cells. Inhibition of cyclin D/Cdk4 kinase
activity in the absence of any change in the expression of either the
cyclin or Cdk subunit strongly suggests that a cyclin kinase inhibitor
is involved in the response. The effects of dexamethasone appear to be
mediated by induction of p21Cip1, which increases 5- to
10-fold after addition of glucocorticoids to mid-log phase cells. There
is a corresponding increase in the abundance of ternary complexes that
contain Cdk4 and p21, and these ternary complexes either lack or have
very little Cdk4 kinase activity.
Expression of the Cip1 locus is rapidly induced in
glucocorticoid-treated cells. The abundance of p21 mRNA increases >5
fold within 2 h after addition of dexamethasone. In wild type L929
cells, the glucocorticoid receptor is down-regulated after addition of
dexamethasone, and one observes that p21 mRNA expression begins to
decrease about 12 h after addition of the steroid. Nevertheless,
the abundance of p21 protein remains relatively constant for at least
24 h, suggesting that several mechanisms probably impinge on p21
expression in glucocorticoid-treated cells. Induction of p21 mRNA is
associated with an increase in nuclear run-on transcription of
Cip1. We have been unable to detect any change in p21 mRNA
degradation in glucocorticoid-treated cells, although one must be
cautious when interpreting mRNA degradation data obtained by the use of
nonspecific inhibitors such as actinomycin D. The simplest explanation
of the data is that glucocorticoids activate transcription of
Cip1, and p21 mRNA increases thereafter. The effect is
totally dependent upon expression of functional glucocorticoid
receptors, and induction of p21 mRNA can be demonstrated in the
presence of nonspecific inhibitors of translation. The rapidity of the
response, the requirement for glucocorticoid receptor, and the
insensitivity to cycloheximide argue that activation of Cip1
is due to a direct or primary interaction between the Cip1
promoter and the glucocorticoid receptor. Experiments are currently in
progress to identify glucocorticoid response elements within the
Cip1 promoter, but preliminary data indicate that no such
element exists within 3.4 kb upstream of the transcriptional start
site.
The data presented above indicate that glucocorticoids induce
transcription of Cip1. The consequent increase in p21
protein abundance results in a decrease in cyclin D/Cdk4 kinase
activity and a concomitant inhibition of pRb phosphorylation.
Inhibition of pRb phosphorylation accounts, at least in part, for
glucocorticoid inhibition of fibroblast proliferation. It is
interesting to note that glucocorticoids do not cause G0 arrest in L929
cells. Instead, G1 progression is attenuated at some point in mid- to
late G1 (5, 73). Cells escape from this pause point, enter S phase, and
complete cell division with a frequency that is sufficient for a
population-doubling time of >100 h. If one assumes that a certain
degree of Rb phosphorylation much be achieved before S phase can be
initiated in normal fibroblasts, then one must assume that
phosphorylation of pRb increases slowly at this pause site, to the
extent that the protein ultimately accumulates the appropriate number
of types of phosphorylations to permit G1 progression and entry into S
phase. In order for such phosphorylation events to accrue slowly, the
rate of dephosphorylation of pRb during G1 must be very low, so that
even very low levels of pRb kinase activity can, given sufficient time,
ultimately achieve critical phosphorylation of the tumor suppressor
protein. The biochemistry of pRb phosphorylation is very complex and
poorly understood, and less is known about dephosphorylation of these
key regulators of G1 progression. It is clear, however, that these
processes will prove to be central to hormonal control of cell
division, and our understanding of how hormones regulate the commitment
to DNA replication must ultimately be understood in terms of the
phosphorylation state of pRb.
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MATERIALS AND METHODS
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Cell Culture
L929.06 cell line is a subclone of the mouse L cell (NCTC 929)
selected for its sensitivity to dexamethasone (71). The L cell clone
designated E82.A3 is a null variant isolated and characterized in
Housleys laboratory (55). W. V. Vedeckis laboratory engineered
E82.A3 to express the mouse glucocorticoid receptor from a stably
integrated tetracycline repressible expression vector, and a detailed
description of these cells will be published elsewhere (P. Wei, Y. I.
Ahn, P. R. Housley, J. Alam, and W. V. Vedeckis, manuscript submitted).
L cells were grown in DMEM supplemented with 5% FBS and subcultured by
treatment with trypsin.
Reagents
The anti-p21 antibody used in Western blotting was obtained from
Dr. David Hill of Oncogene Science Inc. (Cambridge, MA). All other
antibodies (including anti-p21 antibody used for immunoprecipitation)
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Recombinant GST-Rb protein was prepared from Escherichia
coli extracts, as previously described (8).
Western Blotting and Immunoprecipitation
Immunoblotting was carried out essentially as described by Rhee
et al. (9). Briefly, cells were harvested by trypsinisation,
collected by centrifugation (at 4 C), and lysed for 15 min on ice in
lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl,
0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, with 10 µg/ml each
of aprotinin, phenylmethylsulfonyl fluoride, leupeptin, and pepstatin
added just before use). Cell lysates were cleared by centrifugation (in
a microfuge at maximum speed for 15 min at 4 C), and protein
concentration was determined by the method of Bradford (74). Samples
were denatured by boiling for 5 min in 2x sample buffer containing
0.125 M Tris-HCl, pH 6.8, 1% SDS, 20% glycerol, and 10%
ß-mercaptoethanol. Samples (60 µg/lane) were separated by SDS-PAGE
on 12% polyacrylamide gels using a mini gel apparatus (Bio-Rad,
Richmond, CA). Proteins were then transferred onto nitrocellulose
membrane (Schleicher & Schuell, Keene, NH) using a semidry transfer
apparatus (Bio-Rad). Membranes were incubated overnight at room
temperature in blocking solution (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.05% Tween 20, 5% nonfat dry milk),
followed by a 3-h incubation at room temperature in primary antibody
diluted in blocking solution. Membranes were washed three times in
blocking solution, followed by a 1-h incubation in horseradish
peroxide-conjugated secondary antibody diluted in blocking solution.
Membranes were washed three times in blocking solution before the
specific protein bands were detected by the enhanced chemiluminescence
system (Amersham, Arlington Heights, IL).
Immunoprecipitation was undertaken by the same method as above except
that before separation of the proteins by SDS-PAGE, equal amounts (150
µg) of protein were incubated with indicated antibody for 2 h on
ice with occasional mixing. Antigen-antibody complexes were recovered
by incubation for 2 h at 4 C with 30 ml Protein A-Sepharose beads
(0.1 g suspended in 1 ml lysis buffer) (Sigma, St. Louis, MO), washed
four times in lysis buffer, and dissolved in 2x sample buffer followed
by separation and detection as described above.
Rb Kinase Assay
Phosphorylation of recombinant GST-Rb was assayed as described
by Rhee et al. (8). Approximately 105 cells were
washed in PBS and lysed by adding 100 µl IP buffer (50 mM
HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10
mM ß-glycerophosphate, 1 mM NaF, 10%
glycerol, 0.1 mM sodium orthovanadate, 1 mM
dithiothreitol (DTT), plus 10 µg each of aprotinin, leupeptin,
pepstatin, and phenylmethylsulfonyl fluoride added freshly), followed
by sonication of three 15-sec bursts with cooling on ice in between.
The sonicated mixtures were clarified by centrifugation at 12,000
x g at 4 C. The protein content of the supernatant fraction
was measured, and 500 µg of protein were transferred to a
microcentrifuge tube along with 1 µl of cyclin D or Cdk4 polyclonal
antibody. This mixture was incubated for 12 h on ice with occasional
mixing. Fifty microliters of protein A-Sepharose beads were added to
the mixture and incubated for 23 h at 4 C with rocking. The beads
were sedimented by centrifugation for 30 sec at 4 C at 10,000 x
g and washed three times with IP buffer and twice with 50
mM HEPES, pH 7.5, containing 1 mM DTT. Twenty
two microliters of kinase buffer containing 1.5 µg recombinant
GST-Rb, 10 mCi [
-32P]ATP (6000 Ci/mmol), 50
mM HEPES, pH 7.5, 10 mM MgCl2, 2.5
mM EGTA, 10 mM ß-glycerophosphate, 1
mM sodium fluoride, 1 mM DTT, and 0.1
mM sodium orthovanadate were added to the beads. The
reaction mixture was incubated at 30 C for 30 min. The reaction was
stopped by adding 25 µl of 2x SDS-PAGE sample buffer, and the
samples were processed for electrophoresis on a 6% polyacrylamide gel.
The gel was dried onto a Whatman 3MM paper (Clifton, NJ) and
autoradiography carried out.
Cip1 Gene Expression
Analysis of p21 mRNA abundance was carried out by Northern
blotting (5, 6, 7). Mouse Cip1 cDNA was labeled and used as a probe.
Nuclear run-on transcription was assayed using published procedures
(5, 6, 7). Mouse Cip1 cDNA was used to measure Cip1
transcription, and transcription of 5S RNA was measured as an internal
control. All autoradiographic and chemiluminescent data were analyzed
using a Lynx 5000 digital image analysis system with version 5.5
software. Exposures were controlled so as to provide a linear response
range of at least 5-fold.

View larger version (28K):
[in this window]
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|
Figure 9. Nuclear Run-on Transcription of Cip1
in Glucocorticoid-Treated Cells
Mid-log phase cultures of E92T4/GR3 cells were grown in the absence or
presence of 0.1 µg/ml tetracycline. Cultures were treated with
dexamethasone for 2 h, and parallel cultures were untreated.
Nuclei were isolated and transcription was carried out in the presence
of [ 32P]UTP. Labeled RNA was extracted and hybridized
to Cip1 cDNA and 5S RNA genes immobilized on nitrocellulose filters, as
described in Materials and Methods. Hybridization was
measured by densitometry, and data were normalized to transcription of
5S RNA genes.
|
|
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to express particular thanks to Ping Wei and
Wayne Vedeckis for providing the tetracycline-repressible GR expression
clone E82T4/GR3 and to Wade Harper for providing mouse Cip1 cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: E. Aubrey Thompson, Ph.D., Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-0645.
This work was supported in part by Grant P01-AG10514 from the National
Institute of Aging and Grant R37-CA24347 from the National Cancer
Institute.
Received for publication April 15, 1996.
Revision received January 30, 1997.
Accepted for publication February 5, 1997.
 |
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