(Received for publication, June 12, 1995; and in revised form, August 3, 1995)
From the
The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.
Breast cancer is one of the most prevalent of all cancers and is
characterized by hormonal growth control. The proliferation and
phenotype of breast cancer cells is determined to a great extent by
estrogen, and elevated concentrations of estrogen may contribute to an
increase in the risk of breast cancer(1) . Because of the
hormonal dependence of breast cancer, the presence of estrogen receptor
(ER) ()and progesterone receptor (PgR) in breast tumors is
used to predict those patients who will benefit from hormonal
therapy(2, 3, 4, 5) . Although
significant concentrations of estrogen receptor have been detected in
approximately 60% of human breast cancers, only 60% of these
ER-positive tumors respond to endocrine
therapy(2, 3, 4, 5) . Absence or
loss of estrogen responsiveness in breast tumors correlates with a more
malignant form of the disease. Several mechanisms may be responsible
for the loss of estrogen responsiveness in human breast cancer;
including the loss of ER expression, the presence of subpopulations of
cells which are ER negative, and the presence of ER variants with
altered activity. Several ER variants, including point mutations and
alternately spliced forms, have been described in tumors and in breast
cancer cell lines (6, 7) . Some of these variants lack
the ability to bind estradiol, whereas other variants are active in the
absence of ligand. In addition to the above mechanisms, treatment of
breast carcinoma cell lines with the anti-estrogen tamoxifen has been
shown to result in the accumulation of ER that has lost its ability to
bind estradiol(8) . Phosphorylation of the estrogen receptor
has also been shown to regulate its function. In the case of hormone
binding, it has been suggested that dephosphorylation of a tyrosine
residue in the hormone binding domain of the receptor blocks estradiol
binding(9) . Interactions with other factors have also been
shown to modulate steroid receptor function. The unregulated expression
of c-Jun or c-Fos proteins inhibits ER activity in human breast
cancer-derived cells(10, 11) .
Several lines of evidence suggest that protein kinase C may play a role in the loss of estrogen responsiveness in breast cancer. In human mammary carcinoma cells, an inverse relationship exists between the levels and activity of protein kinase C and the level of estrogen receptor(12) , that is, the activity of protein kinase C is several times higher in ER-negative breast cancer cell lines than in ER-positive cell lines(13) . We and others have also shown that treatment of the breast carcinoma cell line MCF-7 with TPA, an activator of protein kinase C, results in a decrease in the level of ER (14, 15) . This effect is mediated by a post-transcriptional destabilization of the ER mRNA(14) .
The data presented in this report demonstrate dramatic effects of TPA treatment on ER activity which are independent of its effect on the level of ER. Following long term treatment, TPA inhibits the activity of the ER as shown by the lack of estradiol induction of PgR and the inability of estradiol to activate an estradiol-inducible reporter gene. The inhibitory effect of TPA on ER activity is due to the induction/activation of a factor that blocks the binding of estradiol to the ER.
A whole cell-binding assay was also
used to measure the binding capacity of the estrogen receptor. 80,000
cells per well were plated in 6-well plates. Following treatment with
TPA, cells were washed with PBS and [H]estradiol
(0.1-6.0 nM) was added in the presence or absence of a
100
excess diethylstilbestrol to determine nonspecific binding.
Binding assays were done in triplicate at 37 °C for 1 h. Cells were
washed three times with PBS and disrupted by sonication in Hanks'
solution. Samples were counted, and the data were analyzed by the
method of Scatchard.
Hormone-binding assays
were performed by the charcoal adsorption method (18) . Cytosol
was incubated with [H]triamcinolone acetonide in
an ice water bath overnight in a total volume of 100 µl containing
0.6 mg of protein. Nonspecific binding was determined by incubating the
mixture with 1000-2000-fold molar excess of nonradioactive
triamcinolone acetonide. After 18 h, 300 µl of 2.5% dextran-coated
charcoal were added followed by mixing for 10 s. The tubes were placed
at room temperature for 10 min and then centrifuged for 8 min.
[
H]Triamcinolone acetonide present in the
supernatant was determined using a scintillation counter. Specific
hormone binding was obtained by subtracting nonspecific binding from
total binding. The binding assays were performed in triplicate.
Dissociation constants were determined by computer analysis of the data
based on the Michaelis-Menton equation [HR] =
[H][R]/[H] + K
where HR = hormone-receptor complex, H = free
hormone concentration, R = free and complexed receptor.
For
Western blot analysis, electrophoresis was performed in 12% slab gels
according to Laemmli (19) as described previously by Blackshear (20) . Lysozyme (15.3 kDa), -lactoglobulin (18.3 kDa),
carbonic anhydrase (27.8 kDa), ovalbumin (44.2 kDa), bovine serum
albumin (71 kDa), phosphorylase B (105.7 kDa), and H-chain of myosin
(196 kDa) from Life Technologies, Inc., were used as molecular mass
standard markers. Cytosol was mixed with an equal volume of
SDS-polyacrylamide gel electrophoresis sample buffer, boiled for 4 min,
and loaded onto the gel. After electrophoresis, the gels as well as
nitrocellulose membranes were immersed in transfer buffer (48
mM Tris, 39 mM glycine, pH 9.2, 20% methanol,
0.00375% SDS) for 20 min. Proteins were transferred from gels to
nitrocellulose membranes. The membranes were blocked with 5% non-fat
milk in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl,
0.05% Tween 20) overnight at room temperature on an orbital shaker. The
membranes were washed three times with TBST and incubated with primary
antibody against the glucocorticoid receptor (GR49) at room temperature
for 1 h with shaking. Unbound antibody was removed by washing as
described above. The membrane was then incubated for 1 h with
peroxidase-conjugated rabbit anti-mouse antibody. The unbound antibody
was removed as described above. The membranes were then incubated with
detection solution (Amersham Corp.) for 1 min and exposed to
Hyperfilm
for 2-5 s.
A second transfection assay system was also employed. In these experiments, the reporter plasmid ERE-TK-Luc (31) was used. Cells were plated in 6-well plates in IMEM supplemented with 10% FCS. The next day, cells were transfected with 1 µg of plasmid DNA using Lipofectamine (Promega) in serum-free IMEM. After 5 h at 37 °C in serum-free IMEM, medium supplemented with 10% FCS was added to achieve a final concentration of 5% FCS. The cells were incubated overnight at 37 °C. The next day, the medium was removed, and the cells were washed with PBS. Cells were maintained in phenol red-free IMEM supplemented with 5% CCS for 48 h. Treatment with TPA and subsequent induction with estradiol were performed as described above. After treatment, cells were harvested, and a cellular extract was obtained as described above. The cellular extract was analyzed for luciferase activity using a luminometer.
For the DNA mobility shift assay, a P-labeled
estrogen-responsive element (ERE; 5`-GATCCTCACGGTCACAGTGACCTGCCCGGGATT)
was incubated with nuclear extracts from MCF-7 cells for 20 min at room
temperature in a buffer containing 10 mM Tris, pH 7.5, 100
mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and
6% glycerol. After incubation, 1 µg of antibody to the ER was added
to the appropriate samples. After a 20-min incubation at room
temperature, samples were loaded onto a 6% polyacrylamide gel in 1
TBE (50 mM Tris borate, pH 8.3, and 0.5 mM EDTA) and run at 20 mA/gel at 4 °C. The gel was dried, and the
bands were visualized by autoradiography.
Figure 1:
Effect of TPA on the steady state
level of ER protein and mRNA. MCF-7 cells were grown in IMEM
supplemented with 5% fetal calf serum. When cells were 70% confluent,
the medium was changed to phenol red-free IMEM containing 5% CCS. Cells
were grown in this medium for 2-3 days and then treated with 100
nM TPA. Twenty-four hours later, medium containing TPA was
removed, and the cells were maintained in fresh medium for 3 days. A, the levels of ER protein () and mRNA (
) were
determined by an enzyme immunoassay and a RNase protection assay,
respectively, as described under ``Materials and Methods.'' B, the levels of ER protein (
) and ER binding capacity
(
) were determined simultaneously by an enzyme immunoassay and
a whole cell assay, respectively, as described under ``Materials
and Methods.''
To determine
whether the decrease and subsequent recovery of ER protein corresponds
to a similar change in estradiol-binding sites, the concentration of ER
protein and binding capacity were measured simultaneously (Fig. 1B). The binding of estradiol to the ER was
determined by a whole cell-binding assay using increasing amounts of
[H]estradiol. Following treatment with TPA, a
significant decrease in the estradiol binding capacity was observed.
The number of estrogen-binding sites decreased from 592 fmol/mg of
protein in control cells to 119 fmol/mg of protein in cells treated
with TPA for 24 h. These results are consistent with the results
obtained with the enzyme immunoassay. However, following removal of TPA
from the medium, the number of binding sites did not increase. Although
the concentration of ER protein returned to control values, the number
of estrogen-binding sites remained at approximately 20% of control
values.
The discrepancy between the concentration of receptor
protein and hormone binding capacity was unexpected. To confirm that
TPA did indeed induce a decrease in the number of estradiol-binding
sites which did not return to control values upon TPA removal, a number
of conditions were employed in the cytosol-binding assay. To rule out
the possibility that TPA directly interfered with estradiol binding to
its receptor, the ability of the ER to bind
[H]estradiol in the presence of added TPA was
determined. Binding experiments were also performed at 4 and 37 °C.
The data from the different binding assays are summarized in Table 1. Independent of the assay conditions, the level of
[
H]estradiol binding was approximately 20% of the
expected values when the cells were treated with TPA. TPA added to the
binding reaction of untreated cells did not affect hormone binding.
Scatchard analysis of the data indicated that treatment of cells with
TPA did not affect the dissociation constant of the estrogen receptor.
These data indicate that, although the levels of ER protein and ER mRNA
returned to control levels 72 h after the change of medium, the ER was
not capable of binding estradiol.
Figure 2: Effect of TPA on induction of progesterone receptor. MCF-7 cells were grown as described in Fig. 1. Prior to the addition of estradiol, cells were treated with TPA for 24 h (TPA) or treated with TPA for 24 h and allowed to recover in fresh medium for 48 h (R). The level of progesterone receptor protein was determined using an enzyme immunoassay.
The effect of TPA on estrogen receptor activity was also studied in a transient transfection assay with the reporter gene Vit-TK-CAT(27) . In this construct, the thymidine kinase promoter is under the control of an ERE. The level of CAT activity was determined by thin layer chromatography. The data from several independent experiments are summarized in Fig. 3. In control cells, the addition of estradiol produced a 2-fold increase in CAT activity. In cells treated with TPA for 24 h, estradiol failed to induce CAT activity. In cells treated with TPA for 24 h and subsequently maintained in fresh medium for 72 h, no increase in CAT activity was seen following estradiol treatment. These data suggest that the ER, although present in these cells, is not functional. To rule out the possibility that the lack of induction of CAT activity in the latter cells was due to the length of time of the recovery experiment, cells were also maintained in culture for 4 days after transfection, and the induction of CAT activity by estradiol was determined. The longer time course did not diminish the estradiol response.
Figure 3: Effects of TPA on ER activation of an estrogen-responsive reporter gene. MCF-7 cells were transiently transfected with the estrogen-responsive reporter gene, Vit-TK-CAT, as described under ``Materials and Methods.'' Following transfection, the cells were maintained in phenol red-free IMEM supplemented with 5% CCS for 2-3 days. Cells were treated with 100 nM TPA and 1 nM estradiol for 24 h or treated with TPA for 24 h and allowed to recover in fresh medium for 48 h (R) prior to the addition of estradiol. Cells were harvested, and CAT activity was measured. Data represent the mean ± S.D. (n = 3-5).
To show that the effects of TPA were not Vit-TK-CAT vector-specific, a number of transfection experiments also were performed using a second reporter gene ERE-TK-Luc(31) . In MCF-7 cells, transiently transfected with the ERE-TK-Luc reporter plasmid, treatment with estradiol produced a 3-fold increase in luciferase activity from 120 units/mg of protein in control cells to 360 units/mg of protein in the cells stimulated with estradiol. Treatment of cells with TPA for 24 h prior to the addition of estradiol blocked estradiol induction of luciferase activity (data not shown).
Other studies have shown that treatment of MCF-7 cells with TPA activates the estrogen receptor(33) . To explain the discrepancy between our results and previously reported results, we studied the short term effects of TPA on ER activity in transient transfection assays using the Vit-TK-CAT reporter. Treatment of cells with 1 nM estradiol for 3 h resulted in a 2-fold increase in the level of CAT protein. Pretreatment of cells with TPA for 24 h prior to the addition of estradiol blocked the induction of CAT protein. However, simultaneous treatment with TPA and estradiol for 3 h resulted in a greater increase (4-fold) in the level of CAT protein than in cells treated with estradiol alone (data not shown). To determine if the synergistic effect of short term treatment with the phorbol ester was also observed on the expression of an endogenous gene, the effects of TPA and estradiol on pS2 expression were studied 6 h after treatment using a RNase protection assay. Short term treatment with TPA for 6 h resulted in a 20% increase in pS2 mRNA, whereas treatment with estradiol resulted in a 40% increase in the level of pS2 mRNA. However, the combined treatment of TPA and estradiol resulted in a 175% increase in the level of pS2 mRNA (data not shown). These results demonstrate that the effects of TPA on ER activity are time-dependent. The initial effects of TPA are to activate the receptor, whereas the long term effects are to block receptor function.
Alternately spliced mutants of the estrogen receptor have been identified in breast cancer tumors and cell lines(6, 34, 35, 36, 37) . Mutants which have been identified include deletion of exons 2, 3, 5, and 7. To determine if inactivation of estrogen receptor by TPA was due to the induction of an aberrant ER species by alternative splicing, ER mRNA was analyzed by a RNase protection assay using riboprobes for the entire coding sequence (Fig. 4C). In the case of the pOR-C probe, there was no evidence of an alternately spliced product. Deletion of exons would have resulted in the loss of the 652-bp protected fragment and the appearance of smaller protected fragments. Deletion of exon 1 would result in the appearance of a protected fragment of 568 bp; deletion of exon 2 would give protected fragments of 84 and 377 bp; and deletion of exon 3 would result in the appearance of fragments of 275 and 260 bp. The deletion of both exons 3 and 4 would result in the appearance of a protected fragment of 275 bp in the pOR-C lane and a protected fragment of 229 bp in the pOR-B lane. The fragments protected by the pOR-C probe are not consistent with an alternately splice product. They are, however, consistent with truncated forms of the probe (probe not shown). In the case of deletion of exons 5 and 7, alternate splicing would have resulted in the loss of probes pOR-B and pOR-R and the appearance of several fragments of less than 100 bp. No protected fragments of the expected sizes (44, 76, and 90 bp) appeared after TPA treatment (Fig. 4). These data suggest that inactivation of the estrogen receptor was not due to the induction of an aberrant ER species.
Figure 4: Effect of TPA on the structure of ER mRNA. Total RNA was isolated from MCF-7 cells treated with 100 nM TPA for 24 h. Four different riboprobes for the coding sequence of the ER were employed in RNase protection assays. A, total RNA from control cells; B, total RNA from TPA-treated cells; C, map of the ER riboprobes.
Figure 5:
Concentration effect of TPA on the level
of ER protein and binding capacity. MCF-7 cells were treated with TPA,
2.5-100 nM, for 24 h as described in Fig. 1. ER
protein was determined by enzyme immunoassay as described under
``Materials and Methods.'' () The ability of ER to bind
estradiol was determined using the single point analysis as described
under ``Materials and Methods.'' (
) The experiment
was performed in triplicate and repeated with similar
results.
Figure 6: Effects of H-7 and bryostatin on the TPA-induced inhibition of ER activity. MCF-7 cells were grown as described in Fig. 1. Cells were treated with 1 nM estradiol, 2.5 nM TPA, 100 mM bryostatin (BRYO), or 40 µM H-7. Progesterone receptor protein was determined by enzyme immunoassay as described under ``Materials and Methods.'' The results shown are the mean of two experiments.
Figure 7:
Effect of TPA on glucocorticoid receptor
protein and binding capacity. MCF-7 cells were grown as described in Fig. 1and treated with 2.5 nM TPA for 24 h.
Glucocorticoid receptor (GR) hormone binding activity was
determined as described under ``Materials and Methods.'' The
graph shows the specific hormone binding activity of cytosol from
control () and TPA-treated (
) cells. Insert,
cytosol from TPA-treated and control cells was subjected to Western
blot analysis using the glucocorticoid receptor-specific antibody GR49.
The positions of the glucocorticoid receptor, molecular mass markers,
and a nonspecific band (ns) are
shown.
Figure 8:
Effect of TPA on ER binding to an
estrogen-response element. Nuclear extracts were isolated from MCF-7
cells treated with 100 nM TPA for 24 h and subsequently
maintained in fresh medium for 3 days. The ability of ER to bind to a P-labeled ERE was determined in a DNA mobility shift assay
as described under ``Materials and Methods.'' Lane
1, control; lane 2, control plus and anti-ER antibody; lane 3, TPA; lane 4, TPA plus an anti-ER antibody; lane 5, recovery; lane 6, recovery plus an anti-ER
antibody. Several independent experiments were performed with similar
results.
Figure 9:
Effect of TPA on ER binding capacity in
mixing experiments. Nuclear extracts from MCF-7 cells treated with TPA,
2.5 or 100 nM, for 24 h were isolated as described under
``Materials and Methods.'' The ability of these extracts to
inhibit binding of [H]estradiol to the ER in
control extracts was determined by a single point binding analysis. The
results are presented as the percent of the expected binding. The
expected binding is defined as the amount of binding in the individual
extracts prior to mixing. C, control extract; 2C and 3C, 2 and 3 times the amount of control extract; TPA,
extract after treatment with TPA for 24 h; 3TPA, 3 times the
amount of TPA extract; R, extract after treatment with TPA for
24 h followed by a 48-h recovery; 2R, 2 times the amount of R
extract.
The results presented demonstrate that treatment of MCF-7 cells with TPA has a dual effect on estrogen receptor activity. The initial effects of treatment are to enhance ER activity, whereas the long term effects are to block activity of the receptor. The loss of estrogen receptor activity appears to be due to the induction/activation of a factor which interacts with the ER to block binding of estradiol.
Regulation of ER expression in breast cancer cell lines is a complex process involving transcriptional as well as post-transcriptional events controlled by estradiol, anti-estrogens, and activators of protein kinase C(14, 22, 38) . Treatment of MCF-7 cells with TPA results in a decrease in the concentration of ER(14, 15) . This decrease in ER levels is due to post-transcriptional destabilization of ER mRNA(14) . In addition to the effects on the level of ER, TPA has been shown to modulate ER activity in MCF-7 cells. However, these reports are contradictory. It has been shown that TPA treatment of MCF-7 cells transiently transfected with different estrogen-inducible reporter genes results in a synergistic activation of the ER(33) , whereas other reports suggest that TPA and protein kinase C negatively regulate estrogen receptor activity in MCF-7 as well as other cell lines(10, 11) . The results of this study demonstrate an increase in estrogen receptor activity following short term treatment with TPA and an inactivation of the ER following long term treatment with the phorbol ester. The observed differences appear to be due to treatment conditions.
Although the inhibitory activity present in TPA-treated breast cells remains to be identified, c-Jun and c-Fos are possible candidates. Many interactions between the protein kinase C-mediated signal transduction pathway and steroid receptors have been reported(10, 11, 39, 40, 41, 42) . c-Jun and c-Fos have been shown to inhibit ER function(10, 11) . In these studies, loss of estrogen responsiveness was demonstrated by transient transfection assays with reporter genes or by DNA mobility shift assays where ER and c-Jun were mixed in vitro. In contrast to these results, activation of the ovalbumin gene by estrogen receptor has been shown to require the fos/jun complex(41) . The reason for the discrepancy between these reports is not clear but may be due to different transfection systems and methodologies. Inhibition of ER function/activity in the former studies may be due to the high copy number of the transfected plasmids and competition for factors required for efficient transcription. In addition to c-Fos and c-Jun, a protein has been identified in HeLa cells which translocates to the nucleus after TPA treatment(43) . This protein interacts with hsp90 which interacts with the ER; hsp90 has shown to be important for estrogenic activity(44) . The ability of this protein to modulate estradiol binding to the ER remains to be defined.
It has been proposed that estradiol binding to its receptor is regulated by a phosphorylation/dephosphorylation process (for review, see (45) ). In this model, dephosphorylation of the ER inhibits estradiol binding, whereas phosphorylation is require for hormone binding. It has been suggested that a nuclear phosphatase is responsible for the removal of phosphate from the receptor and the subsequent loss of hormone binding(46, 47) . In our study, the addition of the phosphatase inhibitor molybdate to the mixing experiments had no effect on hormone binding, suggesting the inhibitory activity in TPA-treated cells was not due to the induction/activation of a phosphatase.
Treatment of MCF-7 cells with TPA has been shown to inhibit growth and change the morphology of the cells, suggesting a differentiation effect in these cells(48, 49, 50) . This ``differentiation'' may result in a change in the pattern of gene expression in these cells. We suggest that one of these changes is the expression of a factor(s) that interacts with the ER. This factor inhibits hormone binding and consequently inactivates the estrogen receptor. In an osteosarcoma cell line stably transfected with ER, loss of ability of ER to bind estradiol has also been observed(51) . These cells are responsive to estrogenic stimulation at subconfluence but become refractory when confluence is reached. The concentration of ER protein and mRNA are similar in the subconfluent and postconfluent cells. However, a decrease in the number of ER-binding sites and loss of estrogen responsiveness is observed in postconfluent cells.
In conclusion, this study demonstrates that activation of the protein kinase C signal transduction pathway modulates ER activity. In short term treatment, TPA synergistically activates the ER in the presence of estradiol. The mechanism for this effect is unknown. In contrast to the short term effects, long term treatment with TPA inactivates the ER. This effect is due to the induction/activation of a factor that interacts with the estrogen receptor to inhibit both the binding of estradiol to the ER and binding of the ER to its response element. This factor may explain, in part, the role of protein kinase C in the loss of estrogen responsiveness in breast cancer.