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Department of Biochemistry and Molecular Biology, Vincent T. Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007
Address all correspondence and requests for reprints to: Dr. Mary Beth Martin, Lombardi Cancer Center, E411 Research Building, 3970 Reservoir Road NW, Washington, D.C. 20007.
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75%) at 6 h. By 24 h, a small
recovery in the amount of messenger RNA was observed. Transcription
run-on experiments demonstrated a decrease of approximately 70% in the
level of ER gene transcription at 3 h. Transient transfections
using an ER promoter-chloramphenicol acetyltransferase construct
demonstrated that after TGFß1 treatment, chloramphenicol
acetyltransferase activity decreased by 50%, suggesting that TGFß1
inhibition of the ER gene transcription is mediated through the ER
promoter. Although treatment with TGFß1 decreased the ER
concentration, the growth factor had no effect on the activity of ER,
as measured by its effects on estradiol induction of progesterone
receptor and pS2, suggesting that TGFß1 does not inhibit
proliferation of MCF-7 cells by blocking ER activity. |
Introduction |
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ER-positive breast cancer cell lines, such as MCF-7, produce growth factors that may act in an autocrine and/or paracrine fashion to influence the proliferation and responsiveness of breast cancer. It has been suggested that the proliferative effects of steroids in normal and neoplastic tissues, such as breast cancer, may actually be mediated by hormone-induced growth factors (1, 2). By binding to specific membrane receptors, growth factors initiate a complex signal transduction cascade that involves sequential phosphorylation-dephosphorylation reactions (for review, see Ref.3). One consequence of activation of signal transduction pathways may be the regulation of phosphorylation of nuclear transcription factors, such as c-Jun and c-Fos, which, in turn, would influence the interaction between activating protein-1 (Jun-Fos heteromer) and steroid receptors (4).
Normal and transformed human breast epithelial cells in culture express TGFß1 messenger RNA (mRNA) and secrete TGFß into their medium (4, 5, 6). In contrast to other growth factors, exogenous TGFß1 and -2 significantly inhibit the growth of normal breast cells and most breast cancer cell lines in vitro (7, 8), suggesting that endogenous TGFß may function as a negative autocrine factor. However, in tumors there appears to be a positive association between TGFß and advanced stages of breast cancer (5, 9, 10, 11, 12, 13). There is also evidence that tamoxifen inhibition of breast cancer growth in vivo involves an ER-independent induction of TGFß in the surrounding stroma. TGFß is a member of a large family of structurally and functionally homologous polypeptides that bind to a family of cell surface receptors and elicit an array of apparently unrelated cellular responses. Five homologous species have been identified in vertebrates, of which TGFß1, -2, and -3 are synthesized by mammalian cells. Although they are encoded by distinct genes located on different chromosomes, these three isoforms are 70–80% homologous. The three isoforms are often coexpressed and have similar biological activities (14). They are interchangeable in most biological assays.
Many cell lines have high affinity binding sites for TGFß on their surface (15, 16, 17, 18, 19, 20, 21). Membrane receptor labeling assays have identified nine distinct proteins that bind TGFß. The most important receptors are I and II, which are glycoproteins, and receptor III, or betaglycan, which is a lower affinity membrane-anchored proteoglycan. These membrane receptors specifically bind different isoforms of TGFß and are coexpressed in many cells.
It has been suggested that cross-talk between growth factor signal transduction pathways and steroid receptors modulates the response to hormones and, in turn, influences normal and/or aberrant cellular responses (2). To date, most studies have examined the effects of growth-enhancing factors on ER expression. Although it had been previously (1) shown that treatment of breast cancer cells with TGFß1 caused a small decrease in the ER concentration, the mechanism of this decrease was not analyzed. The goal of the present report was to study the effects of TGFß1 on ER expression and to determine the mechanism of regulation of ER gene expression by TGFß1. To achieve this goal, the effects of TGFß1 on the steady state levels of ER protein, mRNA, and gene transcription were measured. In addition, the effects of TGFß1 on ER activity were examined.
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Materials and Methods |
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-32P]ATP (SA, >5000 Ci/mmol) were purchased from
Amersham (Arlington Heights, IL). TGFß1 was obtained from R&D Systems
(Minneapolis, MN), and human fibronectin was purchased from Beckon
Dickinson Labware (Bedford, MA). Calf serum as well as
ammoniumpersulfate, phenol, proteinase K, gel mix 6,
vanadylribonucleosidase, cesium chloride, and guanine isothiocyanide
were obtained from Life Technologies (Gaithersburg, MD). The ER and
progesterone receptor (PR) enzyme immunoassay kits were purchased from
Abbott Laboratories (North Chicago, IL).
Tissue culture
Monolayer cultures of MCF-7 cells were grown in Improved Minimal
Essential Medium (IMEM) supplemented with 5% FCS. When the cells were
80% confluent, the medium was replaced with phenol red-free IMEM (22)
containing 5% charcoal-treated calf serum (CCS). Calf serum was
pretreated with sulfatase and dextran-coated charcoal to remove
endogenous steroids that could interfere with the assay (23). After 2
days in these conditions, the medium was changed into serum-free,
phenol red-free IMEM supplemented with fibronectin, glutamine, HEPES,
trace elements, and transferrin, after which 100 pM TGFß1
and/or 1 nM estradiol was added. Cells were harvested at
the times indicated for the different assays.
Plasmids
The probe for the ER, pOR-300, was constructed by subcloning a
300-bp restriction fragment of pOR3 into the pGem4 polylinker regions
using the restriction enzymes PstI and EcoRI
(24). The genomic clone, corresponding to exon 1, Q7, is a 3-kilobase
EcoRI-SalI fragment subcloned into Bluescribe
M13+ (Stratagene, La Jolla, CA) (24). The clone p36B4 was
constructed by subcloning a 220-bp fragment of 36B4 into the
PstI restriction site of the pGem polylinker (24). In
addition, the clones for pS2 (25), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (26), collagenase 92K (27), and PR (28) were
used. The ER promoter-chloramphenicol acetyltransferase (CAT) vector,
pER128-CAT, was constructed by subcloning 128 bp of the proximal
promoter of the ER gene (-128 to +1 nucleotides) into the
HindIII and XbaI restriction sites of the pCAT
enhancer vector (Promega, Madison, WI).
ER and PR protein assays
For analysis of ER and PR protein levels, MCF-7 cells were
cultured and treated as described above. The concentration of receptor
protein was determined using an enzyme immunoassay kit from Abbott
Laboratories (North Chicago, IL). To obtain total receptor protein, the
cells were homogenized by sonication in a high salt buffer (10
mM Tris, 1.5 mM EDTA, 5 mM
Na2MoO4, 0.4 M KCl, and 1
mM monothioglycerol with 2 mM leupeptin). The
homogenate was incubated on ice for 30 min and centrifuged at
100,000 x g for 1 h at 4 C. Aliquots of the total
extracts were then analyzed according to the manufacturer’s
instructions.
To measure the number of estrogen-binding sites and the dissociation constant (Kd) of the estradiol-ER complex, a whole cell, multiple dose, ligand binding assay was used. Cells were plated in 6-well plates (80,000 cells/well). After treatment, the cells were washed with Hanks’ Balanced Salt Solution (HBSS), then treated with various concentrations of [3H]estradiol and incubated with HBSS medium. All points were performed in triplicate. Specific binding was determined as the difference between total and nonspecific binding (BS = BT - BN). Incubations were performed for 1 h at 37 C. The unbound ligand was removed by washing the cells 3 times with HBSS supplemented with 1 mg/ml BSA and once with HBSS. The cells were disrupted either by sonication or by 3 cycles of freezing and thawing. The radioactivity in each well as well as the total radioactivity for each [3H]estradiol concentration were measured in a ß-counter, and the data were plotted according to the Scatchard equation (29). The protein concentration in each well was determined using the Bio-Rad method (Bio-Rad Laboratories, Richmond, CA) and IgG as a standard. The binding affinity (Kd) and capacity (number of binding sites) were determined, and the saturation curves were obtained plotting BS vs. the concentration of the radiolabeled ligand.
Measurement of ER mRNA
Total cellular mRNA was extracted from MCF-7 cells by
homogenization in 6 M guanidine isothiocyanate lysing
buffer containing 5 mM sodium citrate, 0.1 M
ß-mercaptoethanol, and 0.5% sarkosyl. After centrifugation through a
5.7 M CsCl pad at 100,000 x g for 16
h at 20 C (Beckman SW 40 rotor), the amounts of ER, 36B4, pS2, GAPDH,
PR, and collagenase 92K were determined by a RNase protection assay.
For this analysis, homogeneously 32P-labeled antisense
molecules (cRNA) were synthesized in vitro from pOR-300,
p36B4, pGAPDH, and pS2 using T7 polymerase and from pPR and collagenase
92K using SP6. Sixty micrograms of total RNA were hybridized for 12–16
h to the radiolabeled cRNA. After a 30-min digestion at 25°C with
RNase A, 32P-labeled cRNA probes protected by total RNA
were separated by electrophoresis on 6% polyacrylamide gels. The bands
were visualized by autoradiography and quantified by optical
densitometry. The amounts of ER mRNA, PR mRNA, pS2 mRNA, Rb mRNA, and
collagenase 92K mRNA were normalized to the amount of the internal
control 36B4 (25), which is the complementary DNA (cDNA) of the human
acidic ribosomal phosphoprotein PO (30). 36B4 is constitutively
expressed in the presence of estradiol (24) and phorbol esters (31).
GAPDH mRNA was included in some experiments as a second internal
control.
Cytoplasmic mRNA was isolated using the Nonidet P-40 detergent method as described previously (24). The level of cytoplasmic ER mRNA was determined as described above.
Isolation of nuclei
After treatment with TGFß1, MCF-7 cells were harvested and
resuspended in 5 ml 1.5 M sucrose buffer as previously
described (24). The cells were then homogenized with 10 strokes in a
Dounce homogenizer (Kontes Co., Vineland, NJ) using pestle A. The
homogenate was diluted to 15 ml with 1.5 M sucrose and
centrifuged at 10,000 x g for 20 min at 4 C. The
nuclear pellet was resuspended in 0.5 ml nuclei storage buffer (20
mM HEPES, 75 mM NaCl, 0.5 mM EDTA,
0.85 mM dithiothreitol, 0.125 mM
phenylmethylsulfonylfluoride, and 50% glycerol). The nuclei were
stored at -70 C until the transcription run-on was performed.
Transcription run-on assay
The isolated nuclei were in vitro transcribed by
incubation in the presence of ATP, CTP, GTP, and [32P]UTP
for 1 h at 25 C as previously described (24). The newly
transcribed RNA was isolated by digestion with deoxyribonuclease and
proteinase K. The RNA was then purified using RNazol followed by
chloroform extraction and ethanol precipitation. Isolated RNA was
hybridized to nitrocellulose blots containing cDNAs for ER (Q7), 36B4,
pS2, collagenase 92K, and c-myc (exon 2). The samples were
hybridized for 4 days at 42 C. After hybridization, the nitrocellulose
blots were washed and exposed to x-ray film. Autoradiographs were
analyzed by densitometry, and the background was subtracted. Results
were normalized by comparison with the transcriptional level of
36B4.
Transfection and CAT assays
In the transfection assays, 106 MCF-7 cells were
plated in 100-mm dishes and grown in IMEM supplemented with 10% CCS
for 24 h before transfection. Calcium-phosphate DNA precipitates
containing 5 µg ER promoter-CAT vector, 2 µg ß-galactosidase
vector (32), and 23 µg carrier DNA were prepared, and the cells were
transfected by the method of Chen and Okayama (33). Eighteen hours
after transfection, cells were washed, and the medium was replaced with
phenol red-free IMEM supplemented with 10% CCS. After 24 h in
estrogen-depleted medium, 100 pM TGFß1 was added. Cell
lysates were prepared 6 h after TGFß1 treatment and analyzed for
CAT activity. ß-Galactosidase activity was determined as a measure of
the transfection efficiency. The conversion of
[14C]chloramphenicol to its acetylated forms was
determined by TLC. The plates were scanned by a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA). The amount of CAT activity was
normalized to the amount of ß-galactosidase activity. Results are
expressed as percentage of the control CAT activity.
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Results |
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10% decline), and a greater effect with 100
pM TGFß1 (30% decrease; data not shown). These data
are in good agreement with previous results (34) and suggested that a
concentration of 100 pM TGFß1 was sufficient to observe
an effect on the concentration of ER protein. All subsequent
experiments were performed with this concentration. A time course of
the effect of 100 pM TGFß1 on the concentration of ER
protein is presented in Fig. 1
. Treatment of MCF-7 cells
with TGFß1 resulted in a decline of approximately 30% in total
receptor protein by 6 h. The receptor declined from a
concentration of 413.5 fmol/mg protein in control cells to 289.5
fmol/mg protein in TGFß1-treated cells. The concentration of receptor
remained suppressed for 24 h.
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, and the saturation curves are presented in the insets. The
binding assays indicated a 46% decrease in the number of estradiol
sites from 398 to 193 fmol/mg protein after treatment with TGFß1. The
decrease observed in the ligand binding assay correlates well with the
30% decrease in the concentration of ER protein measured by the enzyme
immunoassay. The dissociation constant of the
[3H]estradiol-ER complex, calculated by linear regression
analysis, was 0.117 ± 0.054 nM (n = 3; r =
0.817) and did not show any significant change after treatment with
TGFß1 (Kd = 0.155 ± 0.028 nM; n =
3; r = 0.913). These Kd values are in good agreement
with the previously reported values for the ER [0.1–0.35
nM (31) and 0.1 nM (35)] and suggest that the
binding affinity of the [3H]E2-ER complex did
not change when the cells were treated with TGFß1. These results
indicate that the TGFß1-induced decrease in estradiol-binding sites
resulted from a change in ER protein concentration and was not due to a
change in the binding affinity of the ER.
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as the ratio of the integrated ER signal to the
integrated 36B4 signal. In this study, TGFß1 treatment of MCF-7 cells
resulted in a maximum suppression of ER mRNA to approximately 30% of
the control value by 6 h, with a small recovery in the amount of
mRNA (40% of control values) observed at 24 h. Except for the
small recovery observed at 24 h, the time course of the effects of
TGFß1 on ER mRNA is similar to that of the effects of estradiol on ER
mRNA (24). The small increase at 24 h may be due to the half-life
of the peptide hormone in the tissue culture medium; similar increases
were observed with other growth factors, such as epidermal growth
factor (our unpublished data). The decrease in ER mRNA was not due to a
nonspecific effect of TGFß1 administration, as indicated by an
approximately 2-fold increase in the amounts of collagenase 92K and
retinoblastoma mRNA (data not shown).
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demonstrate that TGFß1 treatment had a similar effect on the amounts
of cytoplasmic and total ER mRNA. After treatment with TGFß1,
cytoplasmic ER mRNA decreased to approximately 40% of control values
by 6 h, with a small increase observed at 24 h.
Effect of TGFß1 on ER half-life
The effects of TGFß1 on the half-life of ER were also determined
in an attempt to explain the lack of correlation between the changes in
ER protein and mRNA. In these experiments, the inhibitor of protein
synthesis, cycloheximide (40 µg/ml), was used to determine the
half-life of the ER. Cells were treated with TGFß1 for 6 h
before the addition of cycloheximide. The amount of ER was measured
with the enzyme immunoassay. In control cells in serum-free medium, the
half-life of ER was 4.4 ± 0.7 h, whereas in TGFß1-treated
cells, the half-life of ER was 3.7 ± 0.9 h (data not shown).
These results indicate that TGFß1 has no effect on the half-life of
the ER. They also suggest that the lack of a corresponding change in ER
protein and mRNA may be due to an effect of TGFß1 on translation of
the ER transcript.
Effect of TGFß1 on the level of ER gene transcription
To determine whether the TGFß1-induced decrease in ER mRNA was a
transcriptional or a posttranscriptional effect, ER gene transcription
was analyzed with a nuclear run-on assay. Transcription run-on assays
were performed using nuclei isolated from MCF-7 cells treated with
TGFß1. Newly synthesized transcripts were hybridized to cDNA probes
immobilized on nitrocellulose blots. The level of transcription was
determined by autoradiography and quantified by scanning densitometry.
To control for potential artifacts, 36B4 transcription was used as an
internal control, and the relative changes in ER transcription were
normalized to the signal obtained for 36B4. The results from the
transcription run-on assays are shown in Fig. 4. After
treatment with TGFß1, there was a decrease of approximately 75% in
the level of ER gene transcription by 3 h, and the level of
transcription remained suppressed for up to 24 h. As a positive
control, the effects of TGFß1 on pS2, c-myc, and
collagenase 92K were also measured. These genes have previously been
shown to be regulated by TGFß. An increase in the levels of pS2
(2-fold), c-myc (7.8-fold), and collagenase 92K mRNA
(3.5-fold) gene transcription was also observed (data not shown). These
data suggest that TGFß1 decreased ER mRNA by inhibition of ER gene
transcription.
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as a percentage of the control value. After
TGFß1 treatment, CAT activity decreased by about 50%, suggesting
that TGFß1 inhibition of ER gene transcription is mediated through
the ER promoter.
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and 7). The data in Fig. 6 show that treatment with 1
nM estradiol for 24 h resulted in a decrease in ER to
20% of the control value. Treatment with 100 pM TGFß1
resulted in a decrease to 70% of the control value. There was no
further decrease in ER when cells were treated with estradiol and
TGFß1. The data in Fig. 7 demonstrate that treatment with estradiol
resulted in a 5-fold induction of PR. Treatment with TGFß1 had no
effect on PR expression or on estradiol induction of PR. Similar
results were obtained when the effects of TGFß1 and estradiol on the
amounts of ER and PR mRNA were studied (data not shown). In addition to
the effects on ER and PR expression, the effect of TGFß1 on estradiol
regulation of pS2 mRNA expression was measured using an RNase
protection assay. Estradiol treatment resulted in a 2.4 ±
0.3-fold induction (n = 3) of pS2 mRNA. Treatment with TGFß1
resulted in a 1.2 ± 0.2-fold increase (n = 3) in pS2 mRNA
and did not inhibit estradiol induction of pS2 mRNA (2.5 ±
0.7-fold increase; n = 3). When TGFß1 was added 24 or 48 h
before the addition of estradiol, the results were similar.
Pretreatment of the cells with TGFß1 before the addition of estradiol
did not block the estradiol-induced decrease in ER or increase in PR
(data not shown). These results suggest that activation of the TGFß1
signal transduction pathway does not modulate the activity of the
ER.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results presented in this paper demonstrate that treatment of MCF-7 cells with TGFß1 resulted in a decrease in the steady state level of ER mRNA. The decline in ER mRNA to a new steady state level corresponded to a parallel decrease in the level of ER gene transcription. The decrease in ER transcription appears to be a specific response to TGFß1 treatment; the transcription of 36B4 and GAPDH was not altered by the growth factor, whereas the transcriptions of pS2, c-myc, and collagenase 92K increase in response to TGFß1 treatment. Transient transfection experiments suggest that TGFß1 may regulate ER gene expression through the ER promoter. TGFß has been shown to regulate several genes by complex transcriptional mechanisms. It stimulates the expression of its own gene through an activating protein-1-binding site in the human TGFß1 promoter (40). In contrast to positive regulation of gene transcription, negative regulation by TGFß is mediated through a TGFß inhibitory element (TIE) (41), which appears to be conserved in the promoter regions of transin (41), elastin (42), collagenase (43), and several other genes. The TIE binds a Fos-containing nuclear protein complex from TGFß-treated cells (41). Sequence comparison of the TIE DNA-binding sequences with the ER promoter using the Genetics Computer Group program (Madison, WI) suggests the presence of a putative TIE sequence in the ER promoter (GTGATGTTTA). The sequence in the ER promoter identified by the search has the greatest homology to the TIE in the human urokinase gene promoter. The sequences differ by only three nucleotides. The role of the putative TIE in mediating the effects of TGFß1 on ER gene transcription are currently under investigation in this laboratory.
In contrast to the effect on ER mRNA and gene transcription, TGFß1 treatment resulted in only a modest decrease in the ER protein concentration. As previous studies have shown a close correlation between changes in the steady state levels of ER protein and mRNA after treatment with either estradiol or TPA (24, 31), we asked whether TGFß1 influenced the translational or posttranslational regulation of ER. The growth factor had no effect on the half-life of the ER, suggesting that the lack of correlation between the steady state levels of ER protein and ER mRNA after treatment with TGFß1 may be due to regulation of ER translation. One possible explanation for translational control may be an effect of TGFß1 on global protein synthesis due to, for example, changes in the phosphorylation of initiation factors, elongation factors, or aminoacyl-transfer RNA synthetase (for review, see Ref.44). Alternatively, treatment with TGFß1 may specifically influence translation of the ER. The precise mechanism responsible for translational control of the ER is not known, but is currently being studied in the laboratory.
Although treatment with TGFß1 resulted in a decrease in ER expression, the growth factor had no effect on the binding of estradiol to the ER. In this study, the concentration of ER, measured by the enzyme immunoassay, was approximately the same as the number of estradiol-binding sites determined by the ligand binding assay. In addition, there was no difference between the binding affinity of estradiol for the receptor in TGFß1-treated cells and that in control cells. The lack of effect of TGFß1 on ER binding is in sharp contrast to the effects seen with TPA, which also inhibits the growth of MCF-7 cells (45, 46). Treatment of cells with TPA results in the complete loss of ER activity. The loss of ER activity appears to be due to the induction/activation of a factor that interacts with the ER to inhibit both the binding of estradiol to the ER and the binding of the ER to its response elements (47). Unlike the results from the TPA study, the results presented in this study provide no evidence that TGFß1 influences cell growth by modulating ER activity.
In addition to TGFß1, the putative ligand for erbB-2, gp30, decreases the amount of ER mRNA (48). When MCF-7 cells are treated with gp30 the amounts of ER protein and mRNA decrease. The decrease in ER mRNA appears to be due to a decreased transcription of the ER gene. Interestingly, TGFß1 and gp30 treatments have different effects on the transcriptional activity of the ER. Treatment with gp30 inhibits ER induction of PR, pS2, and an estrogen-responsive CAT construct, whereas treatment with TGFß1 has no effect on the estradiol-induced increase in PR or pS2.
In summary, this study demonstrates that treatment of human breast cancer cells with TGFß1 results in a decrease in ER mRNA accompanied by a less dramatic change in the concentration of receptor protein. The mechanism responsible for the decrease in ER mRNA is the inhibition of ER gene transcription. Treatment with the growth factor does not have an effect on the transcriptional activity of the ER.
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Acknowledgments |
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Footnotes |
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2 Supported in part by a fellowship from the NCI (Office of
International Affairs).
Received September 13, 1996.
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, Total ER mRNA; •,
cytoplasmic ER mRNA.
