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INTRODUCTION |
TGF-
1 plays a vital
role in the regulation of cell proliferation, differentiation, and
extracellular matrix re-modeling in various cell types (1, 2).
TGF-
carries out its biological effects through three cell surface
receptors, which are referred to as type I (RI), type II (RII), and
type III (RIII). RI and RII are serine/threonine kinases, and an active
receptor complex consists of two molecules each of RI and RII, which
are essential for TGF-
signal transduction and inhibition of cell
growth (3-6).
One of the crucial roles of TGF-
is the growth inhibition of normal
epithelial cells as well as some cancer cells. Because RI and RII are
necessary for TGF-
-mediated growth arrest, mutational inactivation
of either receptor has been reported to generate TGF-
resistance and
hence the loss of the tumor suppressive function of TGF-
in
cancer cells (7-9). DNA methylation of the RI promoter has been
reported in a subset of gastric cancer cells (10). Another mechanism
for loss of TGF-
-mediated tumor suppression is transcriptional
repression of RII due to decreased binding of stimulatory nuclear
proteins to the RII promoter in keratinocytes and breast cancer cells
(11, 12). RI and RII replacement in cells that lack, or show reduced
levels of, TGF-
receptors led to restoration of TGF-
response and
subsequent reversal of malignancy, as seen in breast and colon cancer
cells (13, 14).
The promoters for RI and RII have been characterized (15, 16). Both RI
and RII promoters lack distinct TATA boxes and are highly GC-rich and
depend on Sp1 transcription factor for the initiation of transcription.
The RI promoter contains four consensus and several putative Sp1 sites,
whereas the RII promoter contains two Sp1 sites. The Sp gene family of
transcription factors consists of four members, which are referred to
as Sp1-Sp4. Whereas Sp1, Sp2, and Sp4 are known to be activators of
gene transcription, Sp3 is generally considered to be a repressor (17).
Sp1 and Sp3 transcription factors recognize the same DNA element and
have similar binding affinities. Sp3 has been shown to repress
Sp1-mediated trans-activation of several genes (18-20).
MCF-7 early passage (MCF-7E) breast cancer cells express RI and RII and
are responsive to the growth inhibitory effects of TGF-
. However,
MCF-7 late passage cells (MCF-7L) lack RII, show reduced levels of RI,
and are TGF-
-resistant. Loss or reduced expression of TGF-
receptors was due to low Sp1 protein levels in MCF-7L cells in
comparison to MCF-7E cells (21, 22). Sp1 deficiency was reversed by
5-aza-2'-deoxycytidine treatment of these cells, leading to the
restoration of TGF-
receptor expression and signal transduction
(12). We now show that in addition to Sp1 deficiency, MCF-7L breast
cancer cells express higher levels of Sp3 than do MCF-7E cells, which
express adequate amounts of both receptors and are consequently
sensitive to TGF-
. Sp3 acts as a transcriptional repressor of
TGF-
receptors. Furthermore, the transient expression of CMV-Sp3
cDNA into TGF-
-responsive MCF-7E cells resulted in
dose-dependent down-regulation of TGF-
receptor
mRNA, thus confirming transcriptional repression by Sp3. Similar
results were also found for TGF-
receptor expression in a colon
cancer cell line designated GEO. Taken together, the previous and
present results in the MCF-7 model system indicate that Sp1/Sp3 ratios
dictate expression versus repression of TGF-
receptors RI
and RII.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
MCF-7E cells were obtained from Dr. Soule
(23), and MCF-7L cells were obtained from the American Type Culture
Collection. Cells were grown in McCoy's 5A medium supplemented with
10% fetal bovine serum (Sigma), amino acids, antibiotics, pyruvate,
and vitamins (Life Technologies, Inc.). GEO colon cancer cells were grown in serum-free medium as described previously (14). Cultures were
maintained at 37 °C in a humidified atmosphere of 5%
C02.
EMSA--
The consensus Sp1 oligonucleotide was end-labeled
using [
-32P]ATP, and electrophoretic mobility shift
assays were performed as described previously (12). Wherever Sp3
antibody was used, the nuclear extracts were incubated with 2 µg of
Sp3 antibody (anti-rabbit, Santa Cruz Biotechnology) for 15 min
on ice prior to the addition of 32P-labeled oligonucleotide.
Western Immunoblot Analysis of Sp3--
Nuclear extracts (5 µg) were obtained from MCF-7E and MCF-7L breast cancer cells
and GEO colon carcinoma cells, and Western analysis was performed as
described previously (12). Rabbit anti-human Sp1, Sp3, and c-Jun
polyclonal antibodies were purchased from Santa Cruz Biotechnology.
Southwestern Blotting--
Southwestern analysis was performed
as described previously (20). Briefly, nuclear extracts were resolved
by 7.5% SDS-polyacrylamide gel electrophoresis and electrophoretically
transferred to a nitrocellulose membrane. Following transfer, the
membrane was blocked overnight with 2.5% (w/v) nonfat dried milk in 25 mM HEPES, pH 8.0, 1 mM dithiothreitol, 10%
(v/v) glycerol, 50 mM NaCl, and 1 mM EDTA. The
membrane was then incubated with [
-32P]dCTP-labeled RI
(
618 bp to the start site) and RII (
274 bp to the start site)
promoter probes and poly(dI·dC) as a nonspecific competitor for
4 h. Later, the membrane was washed with wash buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM
EDTA, and 1 mM dithiothreitol), dried, and autoradiographed.
Transfections and Luciferase Assay--
The RI and RII
promoter-luciferase reporter constructs (RI-Luc and RII-Luc,
respectively) were used to determine RI and RII promoter activities,
respectively (15, 24). The Sp1-dependent thymidine kinase
promoter-Luc (TK-Luc) and non-Sp1-dependent
NF
B-Luc constructs were obtained from Promega and Stratagene,
respectively. The CMV-Sp3 cDNA was described previously (18). The
CMV-Sp3 cDNA vector or CMV control vector without Sp3 cDNA,
along with the constructs mentioned above and a
-galactosidase plasmid for normalization (12), was transiently
transfected into MCF-7E breast cancer cells and GEO colon cancer cells
using the Fugene 6 method (Roche Molecular Biochemicals). The CMV-Sp1
cDNA vector or CMV control vector without Sp1 cDNA, along with
RI-Luc, RII-Luc, NF
B-Luc, and a
-galactosidase plasmid, was
transiently transfected into MCF-7L cells. Cells were harvested at
60 h following transfection, and promoter activities were
determined using a commercial luciferase assay (Luciferase Assay
System, Promega). To determine RI and RII mRNA expression,
transfected MCF-7E cells were harvested at 60 h following
transfection, and RNA was isolated to perform reverse transcriptase-PCR
analysis using RI and RII primers as described below. Total RNA from
CMV-control vector- or CMV-Sp3 cDNA vector-transfected MCF-7E cells
was reverse transcribed into cDNA. PCR analysis was then performed
to determine the RI and RII expression levels in control and CMV-Sp3
cDNA transfected-MCF-7E cells using the respective cDNAs as
templates. It was previously reported that Sp3 could not affect the
activities of Sp1-dependent thymidine kinase promoter (19).
Thus, primers for TK were used to demonstrate the specificity of Sp3
effects on RI and RII expression. Primers for TK were used as an
internal control to normalize the expression levels. A total of 30 cycles of amplification were performed. Primers for RI generate an
865-bp fragment (sense primers, TTG TGG CAC GGT GAG AGT GT; antisense primers, TGC TCC TGG GCT ATT GAA TCA). Primers for RII generate a 1003-bp fragment (sense primers, GCC AAC AAC ATC AAC CAC AAC
ACA; antisense primers, TAG TGT TTA GGG AGC CGT CTT CAG). Primers for
TK generate a 340-bp fragment (sense primers; TTC ATA AGC TAC AGC AGA
GG; antisense primers, TTG ACA TCA GCC TGC TTC TT).
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RESULTS |
Expression of Sp3 Protein--
To determine Sp3 protein expression
levels, Western immunoblot was performed using 5 µg each of nuclear
extracts from MCF-7E, MCF-7L, and GEO cells. Western analysis showed
three protein species of 115, 70, and 68 kDa (Fig.
1). The 68-70-kDa species was the result
of differential internal translation initiation (25). Only the 115-kDa
species has been reported to be biologically active. MCF-7L and GEO
cells showed significantly higher levels of all the three Sp3 isoforms
in comparison to MCF-7E cells, whereas there were no differences in the
c-Jun levels, indicating selectivity of Sp3 modulation.

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Fig. 1.
Western immunoblot analysis of Sp3.
Nuclear extracts (5 µg) from MCF-7E and MCF-7L breast cancer
cells and GEO colon cancer cells were resolved via 7.5%
SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose
membrane, and probed with rabbit anti-human Sp3 and c-Jun antibodies.
The Sp3 antibody recognizes three protein species of 115, 70, and 68 kDa. The 68-70 kDa species result from differential internal
translational initiation.
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EMSAs--
EMSAs were performed using MCF-7E and MCF-7L nuclear
extracts and 32P-labeled consensus Sp1 oligonucleotide to
determine the DNA binding activities of Sp3 (Fig.
2). Enhanced binding of a high mobility complex was observed in MCF-7L nuclear extracts in comparison to MCF-7E
extracts (Fig. 2, lanes 2 and 4). To identify
whether the high mobility complex contains Sp3, the nuclear extracts
were pre-incubated with 2 µg of Sp3 antibody to delete Sp3 protein from the extracts prior to the addition of 32P-labeled
oligonucleotide. The high mobility complex was eliminated by the Sp3
antibody in MCF-7L extracts, confirming that the protein-DNA complex
contains Sp3 (Fig. 2, lane 5). This Sp3 antibody has
previously been reported to cause similar depletion of the protein-DNA
complexes following incubation with endothelial cell nuclear extracts
prior to the addition of 32P-labeled kinase domain receptor
promoter of the vascular endothelial growth factor (26).

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Fig. 2.
EMSA. EMSA was performed using
32P-labeled consensus Sp1 oligonucleotide and nuclear
extracts from MCF-7E and MCF-7L cells. Wherever Sp3 antibody was used,
the nuclear extracts were pre-incubated with 2 µg of Sp3 antibody
prior to the addition of 32P-labeled oligonucleotide.
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Transcriptional Repression of RI and RII by Sp3--
Southwestern
analysis using nuclear extracts from MCF-7E and MCF-7L cells and
radiolabeled RI and RII promoters was carried out to demonstrate Sp3
binding in MCF-7L cells. Higher Sp3 binding to RI and RII promoters was
observed in nuclear extracts from MCF-7L cells, suggesting the
plausible role of Sp3 as a transcriptional repressor of TGF-
receptors (Fig. 3, a and
b).

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Fig. 3.
Detection of Sp3 binding to RI and RII
promoters. Southwestern analysis was performed by resolving MCF-7E
and MCF-7L nuclear extracts using 7.5% SDS-polyacrylamide gel
electrophoresis and probing the nitrocellulose membrane following
protein transfer with radiolabeled RI (a) and RII
(b) promoter probes.
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Effect of Exogenous Sp1 on RI and RII Promoter
Activities--
MCF-7L cells, which lack, or show reduced levels of,
TGF-
receptors, express high levels of Sp3 and low levels of Sp1
protein. To determine whether the modulation of Sp1/Sp3 protein levels leads to induction of RI and RII promoter activities, we transfected varying amounts (0-8 µg) of CMV-Sp1 cDNA into MCF-7L cells.
Western analysis showed increasing amounts of the Sp1 in MCF-7L cells (Fig. 4a). c-Jun was used as a
loading control. Cotransfection of RI and RII promoter-luciferase
reporter constructs with the varying amounts of CMV-Sp1 cDNA
resulted in a dose-dependent increase in the RI and RII
promoter activities, thus suggesting that Sp1/Sp3 ratios control RI and
RII promoter activities (Fig. 4b). Sp1 expression enhanced
Sp1-dependent TK promoter activity but not the
non-Sp1-dependent NF
B-Luc control construct, thus
confirming the specificity of Sp1 effects on the
Sp1-dependent RI, RII, and TK promoters.

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Fig. 4.
Effects of ectopic Sp1 and Sp3 on RI and RII
promoters. a, Western analysis using rabbit anti-human
Sp1 antibody was per-formed on the nuclear extracts of CMV-Sp1
cDNA-transfected MCF-7L cells. C-Jun was used as a loading control.
b, MCF-7L cells were transiently cotransfected with CMV-Sp1
cDNA along with RI-Luc promoter reporter, RII-Luc promoter
reporter, TK-Luc promoter reporter, and control NF B-Luc constructs
as described under "Experimental Procedures." Cells were harvested
at 60 h following transfection, and luciferase activity was
determined and presented as relative units. c, West ern analysis using rabbit anti-human Sp3 antibody was performed
on the nuclear extracts of CMV-Sp3 cDNA-transfected MCF-7E cells.
C-Jun was used as a loading control. d, MCF-7E cells were
transiently cotransfected with CMV-Sp3 cDNA along with RI-Luc
promoter reporter, RII-Luc promoter reporter, TK-Luc promoter reporter,
and control NF B-Luc constructs, and (e) GEO cells were
transiently cotransfected with CMV-Sp3 cDNA along with RI-Luc
promoter reporter, RII-Luc promoter reporter, and control NF B-Luc
constructs as described under "Experimental Procedures." Cells were
harvested at 60 h following transfection, and luciferase activity
was determined and presented as relative units. RLU,
relative luciferase units.
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Effect of Exogenous Sp3 on RI and RII Promoter
Activities--
MCF-7E cells, which express TGF-
receptors, contain
high levels of Sp1 and low levels of Sp3. Consequently, we transfected varying amounts (0-8 µg) of CMV-Sp3 cDNA into MCF-7E cells to modulate the Sp1/Sp3 protein levels and evaluated the effects on RI and
RII promoter activities. Western analysis showed increasing levels of
Sp3 in MCF-7E cells (Fig. 4c). c-Jun was used as a loading control. Cotransfection of RI and RII promoter-luciferase reporter constructs with varying amounts of CMV-Sp3 cDNA showed a
dose-dependent decrease in the RI and RII promoter
activities, thus confirming that Sp1/Sp3 ratios control RI and RII
promoter activities (Fig. 4d). The transfection efficiencies
at 0, 2, 4, and 8 µg of CMV-Sp3 cDNA were 100, 86.4, 84.6, and
85.2%, respectively, for the RI-Luc construct and 100, 80.3, 81.1, and
82.4%, respectively, for the RII-Luc construct. Sp3 expression did not
affect the activity of Sp1-dependent TK-Luc and the
non-Sp1-dependent NF
B-Luc control construct, thus
confirming the specificity of Sp3 effects on RI and RII promoter
activities. Although GEO cells express significant levels of Sp3 (Fig.
1), low levels of RI and RII can still be detected in these cells.
Consequently, we determined the effect of ectopic Sp3 expression on the
RI and RII promoter activities in GEO cells as well. Sp3 expression
down-regulated RI and RII promoter activities, indicating that Sp3 acts
as a transcriptional repressor of TGF-
receptors in GEO colon cancer
cells as well (Fig. 4e).
DNA Binding Activities of Transiently Expressed Sp3--
To
determine whether transiently expressed Sp3 was binding to RI and RII
promoters, Southwestern analysis was performed using radiolabeled RI
and RII promoter fragments exposed to nuclear extracts from
CMV-Sp3-transfected MCF-7E cells as well as CMV-control vector-transfected MCF-7E cells. Transiently expressed Sp3 bound to RI
promoter as well as RII promoter (Fig. 5,
a and b).

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Fig. 5.
DNA binding activities of transiently
expressed Sp3. Southwestern analysis was performed by resolving
CMV-control vector- as well as CMV-Sp3 cDNA-transfected MCF-7E cell
nuclear extracts using 7.5% SDS-polyacrylamide gel electrophoresis and
probing the nitrocellulose membrane following protein transfer with
radiolabeled RI (a) and RII (b) promoter
probes.
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Effect of Exogenous Sp3 on RI and RII Expression--
To evaluate
whether transiently expressed Sp3 binding to RI and RII promoters in
MCF-7E cells results in down-regulation of RI and RII transcripts,
reverse transcriptase-PCR analysis using RI and RII primers was
performed on total RNA from varying amounts (0-8 µg) of CMV-Sp3
cDNA-transfected as well as CMV-control vector-transfected MCF-7E
cells. The transfection efficiencies at 0, 2, 4, and 8 µg of CMV-Sp3
cDNA were 100, 80.1, 78.9, and 79.4%, respectively. Sp3
dose-dependent repression of RI and RII expression levels was observed, thus confirming the role of Sp3 as a
transcriptional repressor of TGF-
receptors RI and RII. However, Sp3
transfection did not affect thymidine kinase mRNA expression.
Because the thymidine kinase promoter contains an Sp1 site important
for transcription of the gene, this result indicated that Sp3 did not
affect this site, thus reflecting the selective repression of TGF-
receptors by Sp3 (Fig. 6).

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Fig. 6.
RI and RII expression levels in CMV-Sp3
cDNA-transfected MCF-7E cells. Total RNA from CMV-control
vector-transfected MCF-7E cells or from varying amounts of
CMV-Sp3 cDNA vector-transfected MCF-7E cells were
reverse-transcribed into cDNA, and PCR analysis was performed using
primers for RI, RII, and TK as described under "Experimental
Procedures."
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DISCUSSION |
Loss or reduced expression of TGF-
receptors has been
implicated in TGF-
resistance leading to tumor formation and
progression (8, 9, 13, 14, 27, 28). Whereas MCF-7E breast cancer cells
express TGF-
receptor RI as well as RII and are responsive to growth
inhibition by TGF-
, MCF-7L cells lack RII and show reduced levels of
RI. MCF-7L cells are TGF-
-resistant. In addition, MCF-7L cells were
found to be tumorigenic, and RII replacement reduced malignancy in
athymic nude mice (13). The RI and RII promoters lack distinct TATA
boxes but do contain multiple GC elements and depend on Sp1 for the
initiation of transcription (15, 16). Loss or reduced expression of
TGF-
receptors in MCF-7L cells in comparison to MCF-7E cells was
related to low expression levels of Sp1 transcription factor (22).
However, in this report we present data suggesting that another member of the Sp gene family, Sp3, which has GC element binding affinities similar to those of Sp1, acts as a transcriptional repressor of TGF-
receptors RI and RII.
Sp gene family members Sp1, Sp2, and Sp4 are generally known as
activators of gene transcription, whereas Sp3 is considered to be a
repressor of gene transcription (17). MCF-7L cells express higher
levels of Sp3 in comparison to MCF-7E cells (Fig. 1). The 115-kDa Sp3
protein is the biologically active form, and the inactive 68-70-kDa
species present in Western blots arise as a result of differential
internal translational initiation (25). However, as opposed to direct
promoter repression, the Sp3-derived 68-70-kDa species can bind GC
elements and thus act as inhibitors of Sp1-mediated gene activation.
Electrophoretic mobility shift assays revealed the DNA-binding
activities of Sp3 from MCF-7L nuclear extracts (Fig. 2). Southwestern
analysis confirmed the binding of the 115-kDa nuclear Sp3 protein to
the RI promoter as well as the RII promoter in MCF-7L cells (Fig. 3,
a and b), which lack RII expression and show
reduced levels of RI. Significantly, nuclear Sp3 binding to the RII
promoter in MCF-7E cells was not observed, and only a trace of Sp3
bound to the RI promoter was found (Fig. 3, a and b). Taken together, the Southwestern data from MCF-7E and
MCF-7L nuclear extracts indicate a possible role for Sp3 in
transcriptional repression of TGF-
receptors RI and RII in MCF-7L cells.
The Sp1 dose-dependent up-regulation of RI and RII
promoter-luciferase reporter activities after cotransfection with
varying amounts of CMV-Sp1 cDNA into Sp1-deficient MCF-7L cells
suggested that Sp1/Sp3 protein levels/activities control RI and RII
promoter activities (Fig. 4b). This was confirmed by the Sp3
dose-dependent down-regulation of RI and RII
promoter-luciferase reporter activities after cotransfection with
varying amounts of CMV-Sp3 cDNA into Sp3-deficient MCF-7E cells
(Fig. 4d). Southwestern analysis confirmed that the
transiently expressed Sp3 bound to the RI and RII promoters in MCF-7E
cells (Fig. 5, a and b). Most significantly, Sp3
binding to the RI and RII promoters in CMV-Sp3 cDNA-transfected
MCF-7E cells resulted in repression of RI and RII mRNA expression,
as indicated by the reverse transcriptase-PCR analyses (Fig. 6). However, thymidine kinase, which was used as a control, was not affected. It was previously reported that Sp3 could repress the activity of multiple Sp1 sites contained in the dihydrofolate reductase
promoter but not the single Sp1 site contained in the thymidine kinase
promoter (19). The RI promoter contains four consensus Sp1 sites,
whereas the RII promoter contains two (15, 16). Ornithine decarboxylase
promoter activity was also repressed by Sp3 (20). However, Sp3 was able
to trans-activate c-fos and c-myc
promoters (29). Hence, the availability of specific coactivators, corepressors, or other transcription factors may dictate whether Sp3
activates or inhibits transcription of a specific gene. Moreover Sp3
effects may also depend on the context as well as the number of Sp1
binding sites.
It has been reported previously that MCF-7E cells express high levels
of Sp1 in comparison to MCF-7L cells (21, 22). Our present data
indicate that MCF-7E cells express low levels of Sp3 in comparison to
MCF-7L cells (Fig. 1). Consequently, the high Sp1/Sp3 ratios in MCF-7E
cells appear to support RI and RII expression, whereas low Sp1/Sp3
ratios contribute to receptor repression in MCF-7L cells. This
hypothesis was confirmed by the ectopically driven increase of Sp1/Sp3
ratios in MCF-7L cells by CMV-Sp1 cDNA expression in MCF-7L cells
and the decrease of Sp1/Sp3 ratios in MCF-7E cells by Sp3 cDNA
expression, which resulted in repression of RI and RII expression (Fig.
6). The correlation between Sp1/Sp3 ratios and expression of RI and RII
was also observed in GEO human colon carcinoma cells. Thus,
transcriptional control of TGF-
receptor expression is dependent
upon the ratio of Sp1/Sp3, and cancer cells can gain a growth advantage
by favoring receptor repression through a combination of reduced Sp1
and elevated Sp3 expression.