From the Free Radical & Radiation Biology Program,
Department of Radiology, and ¶ Holden Comprehensive Cancer Center,
University of Iowa, Iowa City, Iowa 52242
Received for publication, October 24, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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Manganese superoxide dismutase
(Mn-SOD) is a primary antioxidant enzyme whose expression is
essential for life in oxygen. Mn-SOD has tumor suppressor activity in a
wide variety of tumors and transformed cell systems. Our initial
observations revealed that Mn-SOD expression was inversely correlated
with expression of AP-2 transcription factors in normal human
fibroblasts and their SV-40 transformed counterparts. Thus we
hypothesized that AP-2 may down-regulate Mn-SOD expression. To examine
the functional role of AP-2 on Mn-SOD promoter transactivation we
cotransfected AP-2-deficient HepG2 cells with a human Mn-SOD
promoter-reporter construct and expression vectors encoding each of the
three known AP-2 family members. Our results indicated that AP-2 could
significantly repress Mn-SOD promoter activity, and that this
repression was both Mn-SOD promoter and AP-2-specific. The three AP-2
proteins appeared to play distinct roles in Mn-SOD gene regulation.
Moreover, although all three AP-2 proteins could repress the Mn-SOD
promoter, AP-2 Manganese superoxide dismutase
(Mn-SOD)1 is a
nuclear-encoded mitochondrial enzyme that catalyzes the first step in
detoxification of O Activator protein-2 (AP-2) is a family of cell type-specific
developmentally regulated transcription factors that have been implicated as critical regulators of gene expression during vertebrate development, embryogenesis, and carcinogenesis (10-12). There are three known members of the AP-2 gene family, AP-2 A role for AP-2 transcription factors in the development of cancer has
become increasingly clear. AP-2 is involved in the development and
progression of human melanoma by altering the regulation of the
c-kit and MCAM/MUC18 genes (19-21). In addition, AP-2
factors are involved in development or progression of the malignant
phenotype of human breast cancer cells. AP-2 has been shown to
participate in the regulation of the important oncogenes erbB-2 and erbB-3 (15, 22, 23), as well as the
cell cycle regulatory gene p21WAF1 (24, 25) and the extracellular
matrix degrading enzyme MMP-2 (26). Thus, the involvement of AP-2
factors in establishing and/or maintaining the cancer phenotype is well established. However, no study to date has focused on the effects of
AP-2 in modulating the important and novel tumor suppressing function
of Mn-SOD.
We previously described a model system that differentially expressed
AP-2 protein and DNA binding activity, the MRC-5 human lung fibroblast
cell strain and its SV40-transformed counterpart MRC-5VA (27).
Interestingly, Mn-SOD expression was inversely correlated with AP-2 Plasmids and Reporter Constructs--
The human SOD2
promoter reporter construct Cell Culture and DNA Transfection--
The normal human fetal
lung fibroblast strain MRC-5 was obtained from American Type Culture
Collection (ATCC). The SV40-transfromed cell counterpart cells,
MRC-5VA, were a kind gift from Dr. Peter Karran. These two cell
cultures were routinely maintained in Dulbecco's minimum essential
medium (Life Technologies Inc., Gaithersburg, MD) supplemented with
10% fetal bovine serum and 100 units/ml penicillin/streptomycin. HepG2
human hepatoma cells were obtained from ATCC and were maintained in
Eagle's minimal essential medium supplemented with 10% fetal bovine
serum and 100 units/ml penicillin/streptomycin. Cell cultures were
grown in 6-well Falcon tissue culture plates. The day before
transfection, MRC-5VA cells were plated at 2-4 × 105
cells/well and HepG2 cells were plated at 4-5 × 105
cells/well. Transient transfections were performed with Superfect transfection reagent for 6 h according to the specifications of the manufacturer (Qiagen). The transfected cells were harvested 2 days
later. CMV- Determination of Transfection Efficiency--
Reporter Gene Assays--
Luciferase activities were determined
using the Luciferase assay system (Promega) and were normalized
relative to
Chloramphenicol acetyltransferase activities were measured by the
conversion of [14C]chloramphenicol into acetyl- and
diacetylchloramphenicol. Twenty microliters of cell extract was
incubated with 1 µg/µl butyryl-CoA (Sigma Chemical Co., St. Louis,
MO) and 200,000 cpm [1,2-14C]chloramphenicol (ICN, Costa
Mesa, CA) at 37 °C for 2-20 h. CAT activities were normalized
relative to RNA Isolation and Northern Blot Analysis--
Total cellular RNA
was isolated from cell cultures by TRIzol reagent following the
manufacturers' instructions (Life Technologies, Inc.) and was
quantified by spectrophotometry. Ten micrograms of total RNA were
electrophoresed on a 1% agarose formaldehyde gel, transferred, and
fixed onto a nylon membrane (DuPont, Boston, MA). The membranes were
then incubated in prehybridization solution (50% formamide, 10 × Denhardt's solution, 10% dextran sulfate, and salmon sperm DNA 200 µg/ml) for 6 h at 42 °C. Radiolabeled human Mn-SOD or
erbB-3 probes were prepared by random-primed labeling (Roche
Molecular Biochemicals) in the presence of [ Western Blots--
Twenty-five micrograms of nuclear extract
(described below) or total cellular proteins isolated from cells were
loaded per well and subsequently separated on 12.5% SDS-polyacrylamide
gels and transferred to nitrocellulose. The membranes were then
immunoblotted with antibodies specific for AP-2 Gel Mobility Shift Assays--
A double-stranded oligonucleotide
(upper strand, 5'-AGCTCAAGCCCGCGGGCTC-3'; lower strand,
5'-TCGAAGAGCCCGCGGGCTTG-3') was end-labeled with
[32P]dCTP by a Klenow fill-in reaction and was used as
probe in gel mobility shift assays. This probe contained a consensus
AP-2 binding site in the context of the human superoxide dismutase 2 (SOD2) promoter from nucleotides
Gel mobility shift assays were performed by incubating 5 µg of
nuclear protein or 10 µg of total cellular protein together with the
32P-radiolabeled oligonucleotide probe in the presence of 1 µg of poly(dI·dC) (Amersham Pharmacia Biotech, Piscataway,
NJ) and 1× gel shift buffer (10 mM Tris, pH 7.5; 50 mM NaCl; 1 mM MgCl2; 0.5 mM EDTA; 0.5 mM dithiothreitol; and 4%
glycerol) at room temperature for 15 min. The binding reactions were
loaded onto a 5% polyacrylamide gel and run at 35 mA for about 40 min
in 1× TBE (90 mM Tris, 90 mM boric
acid, 2 mM EDTA, pH 8.0). The gels were wrapped in plastic wrap and exposed to x-ray film (Kodak) overnight at Statistical Analysis--
Data were evaluated using Systat 9.0 for windows. All means were calculated from three separate experiments,
and error bars represent standard derivations (S.D.). Analysis of
variance-Tukey was used to determine the significance of differences at
p < 0.05.
Transformed Cells Express Lower Levels of Mn-SOD Than Their Normal
Cell Counterparts--
The levels of constitutive Mn-SOD mRNA and
protein expression in normal human lung fibroblast MRC-5 and its
SV-40-transformed counterpart MRC-5VA were compared by northern and
Western blot analyses (Fig. 1). Both the
4- and 1-kb Mn-SOD mRNA transcripts were abundant in MRC-5 cells.
In contrast, the steady-state levels of both the 4- and 1-kb Mn-SOD
mRNA transcripts were significantly decreased in MRC-5VA cells. We
also performed a Mn-SOD Western blot in MRC-5 and MRC-5VA cells. The
steady-state levels of Mn-SOD protein in MRC-5VA cells were also lower
than in MRC-5 cells, consistent with the results of the Northern
blot.
AP-2 Proteins Are Constitutively Expressed in Transformed
Cells--
To determine whether AP-2 proteins were differentially
expressed in the normal and SV40-transformed cell counterparts, we performed Western blots using specific antibodies to each AP-2 family
member. The results from this experiment, shown in Fig. 2, demonstrated that AP-2 family members
AP-2 Endogenous AP-2 Proteins Bind to cis Elements in the Mn-SOD
Promoter Region--
To elucidate the cause for the reduced expression
of human Mn-SOD in MRC-5VA cells, we studied the 5'-flanking region of
the human SOD2 gene, which expresses Mn-SOD. The
SOD2 promoter is composed of a GC-rich region that contains
multiple AP-2 binding sites previously identified by DNA footprinting
(29). To examine whether the AP-2 proteins in MRC-5VA cells could bind
to at least one of these putative regulatory sites, gel mobility shift
assays were performed using 32P-labeled oligonucleotides
5'-AGCTCAAGCCCGCGGGCTC-3', derived from SOD2
promoter at position AP-2 Represses Mn-SOD Promoter Activity--
Because AP-2 and
Mn-SOD expression were inversely associated in MRC-5 and MRC-5VA cells,
we hypothesized that AP-2 was acting as a transcriptional repressor on
the Mn-SOD promoter. To test this hypothesis, we examined whether
expression of AP-2 proteins could suppress Mn-SOD promoter activity in
the AP-2-deficient HepG2 cell line. We first performed Western blots to
determine whether the human AP-2 AP-2 Family Members Transactivate the c-erbB-3 Promoter--
To
determine whether the observed repression was specific to the Mn-SOD
promoter or a general effect of AP-2 in this system, we studied the
effect of AP-2 on erbB-3 gene expression and promoter activity. In contrast to the results obtained for Mn-SOD,
erbB-3 mRNA expression was positively associated with
AP-2 proteins in MRC-5 and MRC-5VA cells (Fig.
6A). Thus we hypothesized that
AP-2 was acting as a transcriptional activator on the erbB-3
promoter. To test this hypothesis, we examined whether expression of
AP-2 proteins could transactivate the erbB-3 promoter in the
AP-2-deficient HepG2 cell line. We cotransfected the human
erbB-3 promoter reporter construct, erbB-3-pGL3,
with each of the AP-2 expression plasmids into HepG2 cells. The results
from this experiment, shown in Fig. 6B, indicated that
AP-2 DNA Binding Activity Is Necessary for AP-2-mediated Repression of
the Mn-SOD Promoter--
To determine which functional domains of the
AP-2 protein were responsible for repression of Mn-SOD promoter
activity, we used two AP-2
To determine the effects of these AP-2 dominant negative mutants on
transactivation of the Mn-SOD promoter, we cotransfected AP-2B or
AP-2 AP-2B Relieves AP-2-mediated Repression of the Mn-SOD Promoter in
Vivo--
AP-2B not only lacks DNA binding activity, but also inhibits
DNA binding of endogenous AP-2 AP-2B Reactivates Mn-SOD Expression in SV40-transformed
Cells--
To determine whether endogenous expression of Mn-SOD could
be affected by the dominant negative AP-2B, we transiently transfected MRC-5VA cells with AP-2B expression vector or empty vector. Twenty-four hours after transfection, total cellular proteins were isolated and
immunoblotted with an anti-human Mn-SOD antibody. Untransfected MRC-5VA
cells and their normal MRC-5 counterparts were used as negative and
positive controls, respectively. Results of this experiment are shown
in Fig. 9B. These results indicate that AP-2B could not only
increase the activity of a transfected Mn-SOD reporter construct but
also reactivate expression of endogenous Mn-SOD. Thus, decreased Mn-SOD
expression in tumors in vivo might be relieved by a dominant
negative AP-2 strategy.
Transcriptional regulation of gene expression by AP-2 plays an
important role in mammalian development, differentiation, and carcinogenesis (10-12, 34-36). AP-2 transcription factors affect the
expression of a number of downstream target genes important to the
establishment, maintenance, and progression of the malignant phenotype
(21, 22). The results from this study have, for the first time,
established a functional role for the three known AP-2 family members
in the down-regulation of the tumor suppressor gene Mn-SOD. We were
able to demonstrate that AP-2-induced transcriptional repression
contributes at least in part to the decreased expression of Mn-SOD in
SV40-transformed human fibroblasts.
Several lines of evidence point to a role for AP-2 in suppressing human
Mn-SOD gene transcription. The GC-rich characteristic of the Mn-SOD
promoter and the existence of several known Sp1 and AP-2 cis-regulatory
elements make it a candidate for regulation by transcription factors
Sp1 and AP-2 (37-39). As shown in this study, Mn-SOD mRNA and
protein are abundant in normal fibroblasts, which have undetectable
levels of endogenous AP-2 proteins. In contrast, Mn-SOD expression is
significantly lower in SV40-transformed fibroblasts, which express
abundant AP-2 proteins. Similar results for Mn-SOD expression in WI38
normal human fibroblasts and WI38-VA SV40-transformed human fibroblasts
have previously been reported (3). Moreover, it has been reported that
an Mn-SOD promoter mutation in some cancer cell lines with low Mn-SOD
level creates a new AP-2 binding site that might function to repress
Mn-SOD expression in those cells (40).
The potential role of AP-2 in regulating Mn-SOD expression was
addressed by DNA binding studies and transfection studies. Gel mobility
shift assay data suggested that the region from The three AP-2 protein family members, AP-2 Although it is evident that all three AP-2 proteins can act as
repressors of the Mn-SOD gene, the repression mechanism remains unclear. Although AP-2 is generally considered to be a transcriptional activator, for example on the erbB-3 promoter as we have
shown here, it has also been shown to negatively regulate the
transcription of several genes, including stellate cell type I collagen
(41), K3 keratin (42), acetylcholinesterase (43), and C/EBP A proposed model for the transcriptional repression of the human
SOD2 gene by AP-2 is shown in Fig.
10. In this model, AP-2, either alone
or associated with a corepressor, is bound to the SOD2
promoter to effectively decrease transcriptional initiation. Upon
interference of AP-2 DNA binding, as elicited by expression of AP-2B,
the repression imposed by AP-2 is relieved and transcription is
enabled. This may include binding of the promoter by other transactivating factors as previously discussed (29).
and AP-2
were more active in this regard than
AP-2
. Transcriptional repression by AP-2 was not a general effect in
this system, because another AP-2-responsive gene, c-erbB-3, was
transactivated by AP-2. Repression of Mn-SOD by AP-2 was dependent on
DNA binding, and expression of AP-2B, a dominant negative incapable of
DNA binding, relieved the repression on Mn-SOD promoter and reactivated Mn-SOD expression in the AP-2 abundant SV40-transformed fibroblast cell
line MRC-5VA. These results indicate that AP-2-mediated
transcriptional repression contributes to the constitutively low
expression of Mn-SOD in SV40-transfromed fibroblasts and suggest a
mechanism for Mn-SOD down-regulation in cancer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, AP-2
, and AP-2
(13). The three AP-2 genes are located at different loci in
human genome. Somatic cell hybrids and in situ hybridization to chromosomes revealed that AP-2
gene is located at human
chromosome 6p22.3-24 (14). AP-2
and AP-2
genes mapped to human
chromosomes 6p12 and 20q13.2, the latter being a region that is
frequently amplified in breast carcinoma (13). The three proteins
differ in their N-terminal transcription activation domains but show high conservation (75-85%) within their DNA binding and dimerization domains. All three proteins are capable of DNA binding and
transcriptional transactivation (15). Gel mobility shift assays have
shown that in vitro synthesized AP-2
, AP-2
, and
AP-2
can bind indistinguishably as homo- or heterodimers to probes
corresponding to the AP-2 binding sites within the c-erbB-2 and human
metallothionein IIA promoters (15, 16). The consensus
sequence for DNA binding by AP-2 is 5'-GCCN3GGC-3' (17),
although a number of sites that are specifically footprinted by AP-2
have been shown to differ from this consensus sequence (18).
expression in this model system. In MRC-5VA cells that express low
Mn-SOD levels, aberrant cytosine methylation in intron 2 was associated
with decreased Mn-SOD expression; however, the Mn-SOD promoter
displayed no genetic abnormalities (deletions, rearrangements,
mutations) that could account for the decreased Mn-SOD expression (28).
These observations suggested that Mn-SOD expression might be regulated,
at least in part, at the level of transcriptional initiation. The
presence of multiple AP-2 motifs within Mn-SOD promoter region
preceding the transcription initiation site (29) suggests that AP-2
proteins play an important role in the function of human Mn-SOD
promoter. Thus, based on our previous findings, we hypothesized that
AP-2 proteins participate in down-regulation of Mn-SOD gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
555 Mn-SOD-pGL3, provided by Dr. Daret
St. Clair (30), consisted of nucleotides
555 to +24 relative to the
transcription start site cloned upstream of the luciferase reporter
gene in pGL3-Basic vector (Promega, Madison, WI). The human
erbB-3 promoter reporter construct (erbB-3-pGL3) was made by fusing the human erbB-3 promoter fragment to a
pGL3-Basic vector. The human erbB-3 promoter fragment
consisting of 1118 nucleotides just upstream of the ATG start codon was
generated by polymerase chain reaction. Dr. Trevor Williams provided
the AP-2
cDNA (31), which we subcloned into the pcDNA3
mammalian expression vector (Invitrogen). The AP-2
expression
plasmid RSVNco
was provided by Dr. Helen Hurst (15). The AP-2
expression vector AP-2
-pcDNA3 was provided by Dr. Ronald J. Weigel (32). The human AP-2B expression plasmid pSG5-AP-2B was provided
by Dr. Michael A. Tainsky (33). The AP-2
cDNA (a mutant form of
AP-2
with a deletion of the transactivation domain encompassing
amino acids 31-117) was provided by Dr. Lubomir Turek and was
subcloned into pcDNA3. The pCMV-
-gal reporter construct
(CLONTECH) was used to determine transfection
efficiencies. A purely AP-2-responsive reporter construct, 12× AP-2-tk
CAT, was used to characterize AP-2 transactivating activity and its
inhibition by the dominant negative AP-2 isoforms. This construct was
made by fusing 12 AP-2-responsive elements of human MtIIA gene
(5'-ACCGCCCGCGGCCCGTCTG-3') to the herpes simplex virus
thymidine kinase promoter in the vector Basic-CAT (Promega, Madison,
WI) that expresses bacterial chloramphenicol acetyltransferase (CAT) as
a reporter gene.
-galactosidase control vector DNA (0.5 µg/plate) was
cotransfected to control for transfection efficiency.
-Galactosidase
activity in cell extracts was measured using
2-nitrophenyl-
-D-galactopyranoside (ONPG, Aldrich
Chemical Co., Milwaukee, WI) as a colorimetric substrate.
Twenty-microliter samples of cell extracts were added to 1.5-ml
microtubes containing 66 µl of ONPG solution (4 mg/ml, dissolved in
100 mM sodium phosphate, pH 7.5) and incubated at 37 °C
for half an hour. Conversion of ONPG to galactose and
o-nitrophenyl was then determined spectrophotometrically at
A420 nm.
-galactosidase activity. Reproducibility was ensured by
transfection in triplicate.
-galactosidase activity. Reproducibility was ensured by
transfection in triplicate.
-32P]dCTP.
The probes were subsequently added to the membrane in the
prehybridization solution, and then the blots were hybridized for
16-24 h at 42 °C. Following hybridization, the membranes were washed in 2× SSC (1× SCC is composed of 0.15 M sodium
chloride, 0.15 M sodium citrate, pH 7.0), 0.5% SDS twice
for 15 min each at room temperature and then washed in 0.1× SSC, 0.1%
SDS solution twice for 15 min each at 68 °C. The membranes were
wrapped in plastic wrap and exposed to x-ray film (Kodak) at
80 °C
for 2-48 h. The membranes were then stripped and reprobed with a
[32P]dCTP-labeled cDNA fragment specific for
glyceraldehyde-3-phosphate dehydrogenase as a control for RNA loading
and transfer.
, AP-2
, or AP-2
(Santa Cruz) or Mn-SOD at final dilutions of 1:1000, followed by
horseradish peroxidase-conjugated secondary antibodies at 1:10,000
(Amersham Pharmacia Biotech). Detection was performed with ECL reagent
(Amersham Pharmacia Biotech).
26 to
14 relative to
the major transcription start site. The core binding site is identical
to the human MtIIa AP-2 site. Nuclear proteins were extracted from the
cells as follows. Cells were scraped into 0.5 ml of cold buffer A (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; and 0.5 mM dithiothreitol), lysed with
20 strokes of a Dounce homogenizer (Kontes Scientific Glassware, Vineland, NJ), and centrifuged for 30 s. After centrifugation, the
supernatants were removed, and the pellets were resuspended in buffer C
(20 mM HEPES, pH 7.9; 25% glycerol; 0.42 M
NaCl; 1.5 mM MgCl2; 0.2 mM EDTA;
0.5 mM phenylmethylsulfonyl fluoride; and 0.5 mM dithiothreitol). These mixtures were placed on ice for 15 min and microcentrifuged for 5 min at 4 °C. Then the supernatants were harvested and diluted 1:6 with buffer D (20 mM HEPES,
pH 7.9; 20% glycerol; 0.1 M KCl; 0.2 mM EDTA;
0.5 mM phenylmethylsulfonyl fluoride; and 0.5 mM dithiothreitol). The protein concentrations of the
extracts were determined with BCA protein assay reagents (Pierce Biochemical).
80 °C.
To assess the specificity of the binding reaction, antibodies
specific to AP-2 were used in gel supershift assays to verify that the DNA binding activity measured was due specifically to AP-2 (Santa Cruz
Biotechnology, Santa Cruz, CA). For gel supershifts, 1 µl of
anti-AP-2 antibody was incubated with each binding reaction for 30 min
before loading onto the gel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mn-SOD expression is down-regulated at the
mRNA and protein levels in SV40-transformed human cells.
A, Northern blot analysis. 10 µg of total mRNA from
each cell line was loaded in each lane as labeled on the figure. The
human Mn-SOD cDNA was random-prime radiolabeled and used as probe.
The arrows denote the 4- and 1-kb Mn-SOD transcripts. The
SV40-transformed human lung MRC-5VA cells displayed fewer Mn-SOD
transcripts than MRC-5 normal cells. Glyceraldehyde-3-phosphate
dehydrogenase was used as loading control. B, Western blot
analysis of 20 µg of total cellular protein from each cell line. The
blot was probed with rabbit antiserum against human kidney
Mn-SOD.
and AP-2
were constitutively expressed in MRC-5VA cells but
not in MRC-5 cells. AP-2
was not detectable in either cell line
(data not shown). These results indicated that Mn-SOD gene expression
was inversely correlated with AP-2 expression in MRC-5 and MRC-5VA
cells.
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Fig. 2.
A family of AP-2 proteins is expressed in
SV40-transformed human lung fibroblast MRC-5VA cells but not their
normal counterpart MRC-5 cells. Western blot analysis of AP-2
protein levels in the human lung fibroblast cell strain MRC-5 and the
SV40-transformed counterpart MRC-5VA. 25 µg of nuclear protein from
each cell population was analyzed. The AP-2 and AP-2
proteins
were expressed at detectable levels in MRC-5VA cells but not in MRC-5
cells.
26 to
14 relative to the major transcription
start site. AP-2 DNA binding activity was abundant in the nuclear
extract from MRC-5VA cells (Fig. 3,
lane 2). Furthermore, AP-2 binding activity was positively
identified by gel supershift analysis with antibodies specific to each
AP-2 family member (Fig. 3, lanes 3-5). Although antibodies
against AP-2
and AP-2
gave robust supershifts, antibody against
AP-2
did not provide convincing evidence that AP-2
is part of the
DNA binding complex. This result is consistent with the absence of
AP-2
protein in the MRC-5VA cells as determined by Western blotting
described above. Finally, whereas AP-2 DNA binding activity was
constitutively high in MRC-5VA cells, AP-2 DNA binding activity was
absent in normal human lung fibroblasts MRC-5 cells that express
abundant levels of Mn-SOD (27).
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Fig. 3.
SV40-transformed MRC-5VA cells exhibit
constitutive DNA binding activity of AP-2 and
AP-2
proteins to an element in the Mn-SOD
promoter. Gel mobility shift and supershift analysis for
characterization of endogenous AP-2 protein DNA binding activities in
MRC-5VA cells. DNA-binding activity of AP-2 protein is abundant in
SV40-transformed MRC-5VA cells. Lane 1, probe only;
lane 2, probe plus MRC5-VA nuclear extract; lanes
3-5, gel supershifts with antibodies specific to each indicated
AP-2 family member.
, AP-2
, and AP-2
expression
plasmids could express their respective AP-2 proteins in HepG2 cells. 2 µg of each expression vector or their empty parent vectors were transfected into cells seeded on a 6-well plate. Western blot analyses
were performed, and abundant AP-2 protein was detected in AP-2
expression vector-transfected cells but not in untransfected or empty
vector-transfected cells (Fig. 4). The
human AP-2 expression plasmids were each then cotransfected with human
SOD2 promoter-reporter construct
555 Mn-SOD-pGL3 (Fig.
5A). The
555 Mn-SOD-pGL3 had a high basal promoter activity in HepG2 cells that was diminished in a
dose-dependent manner by cotransfection with each of the AP-2 expression plasmids (Fig. 5, B-D), although AP-2
was slightly less effective in repressing the Mn-SOD promoter compared
with AP-2
and AP-2
. In contrast, AP-2 family members had no
effect on the luciferase activity in cells transfected with control
vector pGL3-Basic (data not shown). These results are unlikely to be attributable to differences in transfection efficiency, because the
luciferase activity was normalized by cotransfection of a
-galactosidase control vector. We conclude therefore that, although all three AP-2 family members can act as transcriptional repressors, AP-2
and AP-2
appear to play the more functionally important role
at the Mn-SOD promoter.
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Fig. 4.
AP-2 proteins were detectable in HepG2 cells
after transient transfection of each AP-2 expression plasmid.
Western blot analysis of AP-2 protein expression in HpG2 cells after
transfection with AP-2 expression vectors. 25 µg of total cellular
protein from each cell population was loaded. The AP-2 , AP-2
, and
AP-2
proteins were abundant in cells transfected with expression
vector but not in untransfected cells or cells transfected with empty
vector.
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Fig. 5.
Repression of Mn-SOD promoter activity by a
family of AP-2 proteins in HepG2 cells. A, schematic
diagram of the 555 Mn-SOD-pGL3 construct. The putative AP-2 binding
sites are indicated by the shaded ovals. B-D,
human hepatoma HepG2 cells were transiently transfected with the
555
Mn-SOD-pGL3 construct (1 µg) and varying amounts of AP-2
(B), AP-2
(C), and AP-2
(D)
expression plasmid as indicated on each x axis. The
differences in AP-2 DNA amounts were compensated with empty vector for
each expression construct so that the total amount of transfected DNA
was equal in each case. Results shown are the means and standard
deviations from three independent transfection experiments.
, AP-2
, or AP-2
alone could transactivate erbB-3 promoter. However, AP-2
and AP-2
were more
active in this regard than AP-2
. These results indicated that AP-2
was capable of transactivating a different AP-2-responsive gene in this
model system and suggested that AP-2-mediated repression was specific
to the Mn-SOD promoter. Taken together, these results further suggest
that the transactivating and transrepressing functions of AP-2 are
distinct, and may be related to the specific promoter context in which
the AP-2·DNA interaction occurs.
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Fig. 6.
AP-2 transactivates the erbB-3
promoter, and AP-2 is positively associated with
erbB-3 mRNA expression. A,
Northern blot analysis. 20 µg of total mRNA from each cell line
was loaded in each lane as labeled on the figure. The human
erbB-3 cDNA was random-prime radiolabeled and used as
probe. Glyceraldehyde-3-phosphate dehydrogenase was used as loading
control. erbB-3 mRNA was expressed in MRC-5VA cells but
was not detectable in MRC-5 cells. B, schematic diagram of
the human erbB-3-pGL3 construct. The putative AP-2 binding
sites are indicated by the shaded ovals. C, AP-2
proteins transactivate erbB-3 promoter in HepG2 cells. HepG2
cells were transiently transfected with erbB-3-pGL3 (1 µg)
and cotransfected with each human AP-2 expression plasmid or their
empty vector (0.5 µg), respectively, as indicated. Results shown are
the means and standard deviations from three independent transfection
experiments.
variants. One is AP-2B, which is a
naturally occurring alternatively spliced product from the AP-2
gene
(33). AP-2B contains the activation domain of AP-2
and part of the
DNA binding domain but lacks the dimerization domain that is necessary
for DNA binding. The other is an engineered AP-2
mutant, AP-2
(residues 31-117), resulting from excision of a PvuII
fragment including most of exon 2 and the entire transactivation domain
of AP-2
. AP-2
lacks the activation domain of AP-2
but retains
the dimerization and DNA binding domains. To characterize the dominant
negative nature of these AP-2 isoforms, we transiently transfected a
synthetic chimeric AP-2-responsive promoter reporter construct, 12X
AP-2-tk-CAT, which contains 12 AP-2 consensus sequences and is purely
AP-2-responsive (Fig. 7A),
together with 0.5 µg of AP-2 expression plasmid and an equal amount
of either AP-2B or AP-2
expression vector into HepG2 cells. These
two AP-2
variants, despite their own inability to transactivate the
12X AP-2-responsive reporter, both could specifically interfere with
the transactivating activities of each AP-2 family member in a
dominant-negative manner (Fig. 7B).
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Fig. 7.
AP-2B and AP-2
specifically interfere with the transactivating activity of all
three AP-2 family members in a dominant-negative manner in HepG2
cells. A, schematic diagram of a synthetic chimeric
AP-2-responsive CAT reporter construct 12X AP-2-tk-CAT. B,
HepG2 cells were transiently transfected with 2 µg of 12X AP-2-tk-CAT
and 0.5 µg of each of the human AP-2 expression plasmids or empty
vector as indicated (open bars). The cells were
cotransfected with 0.5 µg of AP-2B (solid bars) or AP-2
(hatched bars). The fold increase in activity was calculated
by measuring the percent conversion of acetylated forms of
[14C]chloramphenicol, relative to the control activity in
cells transfected with empty vector. Results shown are the means and
standard deviations from three independent transfection
experiments.
expression vector with
555 Mn-SOD-pGL3 into HepG2 cells. The
results of this experiment, shown in Fig.
8A, indicated that expression
of AP-2
alone significantly repressed Mn-SOD promoter activity, but
AP-2B alone had little effect. To determine the DNA binding ability of
the different AP-2 proteins transfected in the HepG2 cells, gel shift
assays were performed after transfection of each AP-2 expression vector
and empty vector respectively. As shown in Fig. 8B, AP-2
,
AP-2
, AP-2
, and AP-2
were all able to bind DNA, whereas AP-2B
was unable to bind DNA, which is consistent with previously reported
results (33). These results suggested that DNA binding activity of AP-2
proteins is necessary for repression of the Mn-SOD gene expression.
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Fig. 8.
Repression of Mn-SOD promoter activity by
AP-2 is dependent on DNA binding. A, HepG2 cells were
transiently transfected with the 555 Mn-SOD-pGL3 construct (1 µg)
and each of the AP-2 variant expression plasmids (0.5 µg) or empty
vector (0.5 µg) as indicated. AP-2
-(31-117) alone
significantly repressed Mn-SOD promoter activity, whereas AP-2B alone
had minimal repressive effect on the Mn-SOD promoter. Results shown are
the means and standard deviations from three independent transfection
experiments. B, gel mobility shift assays confirmed the DNA
binding status of each of the transfected AP-2 proteins in HepG2 cells.
HepG2 cells were harvested 2 days after transfection of the indicated
expression vector. Ten micrograms of total cellular protein from each
cell population was added into each binding reaction (lanes
2-8). AP-2 extract (Promega) was used as a positive control in
lane 9. NS, nonspecific; FP, free
probe. Each AP-2 protein was capable of binding DNA except AP-2B.
(33). Thus we hypothesized that AP-2B
might perturb the repressive activity of AP-2 protein on the Mn-SOD
promoter. To test that hypothesis we cotransfected the AP-2B expression
vector together with
555 Mn-SOD-pGL3 into AP-2-abundant MRC-5VA
fibroblasts. Expression of AP-2B significantly up-regulated the Mn-SOD
promoter activity in a dose-dependent manner (Fig.
9). This result further confirmed that
AP-2 DNA binding plays a key role in suppressing Mn-SOD expression and
suggested that decreased Mn-SOD expression in vivo might be
relieved by a dominant negative AP-2 strategy.
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Fig. 9.
AP-2B relieves repression of Mn-SOD promoter
activity and reactivates Mn-SOD expression in MRC-5VA cells.
A, MRC-5VA cells were transiently transfected with 555
Mn-SOD-pGL3 (1 µg) and increasing amounts of the AP-2B expression
plasmid as indicated on the x axis. The difference in the
amount of AP-2B DNA transfected was adjusted by using pSG5 empty vector
DNA. Results shown are the means and standard deviations from three
independent transfection experiments. B, MRC-5VA cells were
transiently transfected with 0, 1, or 2 µg of empty vector or AP-2B
as indicated on the x axis. 25 µg of total cellular
proteins were separated by SDS-polyacrylamide gel electrophoresis and
then immunoblotted with antibody specific to human Mn-SOD.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
26 to
14 relative
to the major transcription start site in the Mn-SOD promoter was
capable of binding to a family of AP-2 proteins, suggesting that AP-2
may be involved in binding to Mn-SOD promoter and suppress this gene
expression. Evidence supporting this conclusion comes from
cotransfection of human Mn-SOD promoter reporter construct
555
Mn-SOD-pGL3 with expression vectors encoding each of the known AP-2
family members. In each case, AP-2 expression decreased the Mn-SOD
promoter activity dose-dependently in AP-2-deficient HepG2
cells. Northern blot analysis showed that the levels of Mn-SOD mRNA
were significantly suppressed in the SV40 transformed human fibroblasts
MRC-5VA, which constitutively expressed AP-2
, AP-2
proteins.
Furthermore, specific interference of the DNA binding and
transactivating activity of AP-2 in human fibroblasts by expression of
AP-2B, a dominant-negative inhibitor of AP-2, resulted in increased
Mn-SOD promoter activity and reactivation of endogenous Mn-SOD
expression. These results suggest that AP-2 is functionally involved in
repressing human Mn-SOD expression. The extent to which AP-2 might be
central in suppressing Mn-SOD expression is still not clear. However,
our transient transfection studies with HepG2 cells have shown that
expression of each AP-2 family member can impart to hepatoma cells the
ability to suppress Mn-SOD promoter-driven transgenes, and AP-2B
counteracts this suppression. These data strongly argue that AP-2 plays
an important role in regulating human Mn-SOD gene expression.
, AP-2
, and AP-2
,
despite their similarities, are unlikely to have redundant roles in
development. Analysis of the developmental expression of mouse AP-2
has shown that, although some overlap exists, there are distinct
patterns of AP-2
and AP-2
expression in the developing brain and
face (16). An example of the distinct roles these factors may play was
demonstrated by our studies. We found that AP-2
was less able than
the other family members to activate a chimeric promoter containing
high affinity AP-2 binding sites. In addition, although all three AP-2
proteins could suppress the Mn-SOD promoter, AP-2
and AP-2
were
more active in this regard than AP-2
. In the MRC-5VA cells that
express low levels of SOD, both AP-2
and AP-2
were readily
detectable but AP-2
was not.
(44). In
all these cases, it was proposed that AP-2 functions as a repressor by
displacing or competing with a positive transcription factor that has a
binding site overlaps with or is adjacent to the AP-2 recognition site.
As for Mn-SOD, it is possibly related to the interaction or competition
between two transcription factors Sp1 and AP-2 reported previously. St.
Clair (29) found that Sp1 was an important regulator for the
transcriptional activity of human Mn-SOD promoter, whereas AP-2
competed with Sp1 for binding sites that may regulate promoter
function. Alternatively, however, AP-2 may play a role in establishing
or maintaining higher order chromatin structure. Structural changes in
the Mn-SOD gene have been reported during transcriptional activation
(9). Our studies illustrated that repression of Mn-SOD gene expression
by AP-2 appears to be activation domain-independent and DNA
binding-dependent. An AP-2
variant, AP-2
, which lacks
the activation domain of AP-2, could still significantly repress Mn-SOD
promoter activity. However, another AP-2
variant, AP-2B, which
contains the activation domain, but lacks the DNA binding activity, had
little effect on repressing Mn-SOD promoter activity. Moreover,
inhibiting DNA binding activity of AP-2 by AP-2B could relieve the
repression of Mn-SOD in the transformed cells that express AP-2. Taken
together these findings suggest that AP-2 DNA binding plays a major
role in down-regulating the expression of the tumor-suppressing
Mn-SOD.
View larger version (17K):
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Fig. 10.
Schematic diagram of a proposed mechanism
for AP-2-mediated repression of the SOD2 promoter and
its reactivation by the dominant negative AP-2B. Lightly
shaded ovals, AP-2; large white oval, postulated
corepressor; small black ovals, AP-2B; shaded
circles, positive-acting transcription factors such as Sp1;
bent arrow, transcription initiation site.
In summary, we have demonstrated that a family of AP-2 proteins
represses transcription of the human SOD2 gene and leads to decreased Mn-SOD expression. Our findings add to the body of literature supporting a role for AP-2 in carcinogenesis by demonstrating that a
novel tumor suppressing gene, Mn-SOD, is a target of transcriptional repression by AP-2 family members. Further studies will focus on
determining the mechanism(s) of repression of the SOD2 gene by AP-2 family members and their associated cofactors, as well as
assessing their effects on cell growth, differentiation, and carcinogenesis.
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ACKNOWLEDGEMENTS |
---|
We sincerely thank the following individuals
for the indicated materials. Lubomir Turek provided the chimeric
AP-2-responsive reporter construct and AP-2 cDNA. Trevor
Williams, Helen Hurst, and Ron Weigel kindly provided expression
vectors for AP-2
,
, and
, respectively. Daret St. Clair
graciously provided the human SOD2 promoter reporter
construct. John Koland provided the human erbB-3 cDNA.
Michael Tainsky provided the AP-2B expression vector.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Services Grants CA-73612 (to F. E. D.) and P01 CA-66081 (to L. W. O.) from the NCI.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Neuroscience Therapeutics Dept., Pfizer Global Research & Development, 2800 Plymouth Rd., Ann Arbor, MI 48105.
To whom correspondence should be addressed: Free Radical & Radiation Biology Program, B180 Medical Laboratories, The University of
Iowa, Iowa City, IA 52242. Tel.: 319-335-8018; Fax: 319-335-8039; E-mail: frederick-domann@uiowa.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M009708200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Mn-SOD, manganese
superoxide dismutase;
AP-2, activator protein-2;
CAT, chloramphenicol
acetyltransferase;
tk, thymidine kinase;
CMV, cytomegalovirus;
ONPG, 2-nitrophenyl--D-galactopyranoside;
kb, kilobase(s).
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