A Family of AP-2 Proteins Down-regulate Manganese Superoxide Dismutase Expression*

Chun-Hong ZhuDagger , Yuanhui HuangDagger §, Larry W. OberleyDagger , and Frederick E. DomannDagger ||

From the Dagger  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




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-2alpha and AP-2gamma were more active in this regard than AP-2beta . 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

Manganese superoxide dismutase (Mn-SOD)1 is a nuclear-encoded mitochondrial enzyme that catalyzes the first step in detoxification of O&cjs1138;2 radical anion (1). Mn-SOD is a primary antioxidant enzyme that is critical for maintaining normal cell function and survival. For example, Mn-SOD knockout mice develop cardiomyopathy and exhibit neonatal lethality (2). Mn-SOD has also been shown to play an important role in preventing the development of cancer. Cancers from a variety of cell types and etiologies have been reported to exhibit decreased Mn-SOD activity (3-5). It has been reported that the reduced level of Mn-SOD activity in cancer cells was not due to a defect in the primary structure of Mn-SOD protein nor a deletion of the Mn-SOD gene, but rather was due to the defects in the expression of the gene (6). Enforced overexpression of human Mn-SOD cDNA caused suppression of the malignant phenotype in several tumor cell lines (7, 8). Elevated Mn-SOD activity was also correlated with a loss of metastatic capabilities of tumor cells (3). Thus, although it is clear that Mn-SOD has tumor-suppressing activity, the mechanism(s) underlying it decreased expression in cancer cells remains unknown.

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-2alpha , AP-2beta , and AP-2gamma (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-2alpha gene is located at human chromosome 6p22.3-24 (14). AP-2beta and AP-2gamma 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-2alpha , AP-2beta , and AP-2gamma 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-2alpha have been shown to differ from this consensus sequence (18).

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-2alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids and Reporter Constructs-- The human SOD2 promoter reporter construct -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-2alpha cDNA (31), which we subcloned into the pcDNA3 mammalian expression vector (Invitrogen). The AP-2beta expression plasmid RSVNcobeta was provided by Dr. Helen Hurst (15). The AP-2gamma expression vector AP-2gamma -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-2Delta cDNA (a mutant form of AP-2alpha 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-beta -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.

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-beta -galactosidase control vector DNA (0.5 µg/plate) was cotransfected to control for transfection efficiency.

Determination of Transfection Efficiency-- beta -Galactosidase activity in cell extracts was measured using 2-nitrophenyl-beta -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.

Reporter Gene Assays-- Luciferase activities were determined using the Luciferase assay system (Promega) and were normalized relative to beta -galactosidase activity. Reproducibility was ensured by transfection in triplicate.

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 beta -galactosidase activity. Reproducibility was ensured by transfection in triplicate.

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 [alpha -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.

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-2alpha , AP-2beta , or AP-2gamma (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).

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 -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).

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 -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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



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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.

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-2alpha and AP-2gamma were constitutively expressed in MRC-5VA cells but not in MRC-5 cells. AP-2beta 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-2alpha and AP-2gamma proteins were expressed at detectable levels in MRC-5VA cells but not in MRC-5 cells.

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 -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-2alpha and AP-2gamma gave robust supershifts, antibody against AP-2beta did not provide convincing evidence that AP-2beta is part of the DNA binding complex. This result is consistent with the absence of AP-2beta 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-2alpha and AP-2gamma 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 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-2alpha , AP-2beta , and AP-2gamma 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-2beta was slightly less effective in repressing the Mn-SOD promoter compared with AP-2alpha and AP-2gamma . 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 beta -galactosidase control vector. We conclude therefore that, although all three AP-2 family members can act as transcriptional repressors, AP-2alpha and AP-2gamma 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-2alpha , AP-2beta , and AP-2gamma 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-2alpha (B), AP-2beta (C), and AP-2gamma (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 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-2alpha , AP-2beta , or AP-2gamma alone could transactivate erbB-3 promoter. However, AP-2alpha and AP-2gamma were more active in this regard than AP-2beta . 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.

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-2alpha variants. One is AP-2B, which is a naturally occurring alternatively spliced product from the AP-2alpha gene (33). AP-2B contains the activation domain of AP-2alpha and part of the DNA binding domain but lacks the dimerization domain that is necessary for DNA binding. The other is an engineered AP-2alpha mutant, AP-2Delta (residues 31-117), resulting from excision of a PvuII fragment including most of exon 2 and the entire transactivation domain of AP-2alpha . AP-2Delta lacks the activation domain of AP-2alpha 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-2Delta expression vector into HepG2 cells. These two AP-2alpha 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-2Delta 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-2Delta (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.

To determine the effects of these AP-2 dominant negative mutants on transactivation of the Mn-SOD promoter, we cotransfected AP-2B or AP-2Delta expression vector with -555 Mn-SOD-pGL3 into HepG2 cells. The results of this experiment, shown in Fig. 8A, indicated that expression of AP-2Delta 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-2alpha , AP-2beta , AP-2gamma , and AP-2Delta 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-2Delta -(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.

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-2alpha (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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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-2alpha , AP-2gamma 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.

The three AP-2 protein family members, AP-2alpha , AP-2beta , and AP-2gamma , despite their similarities, are unlikely to have redundant roles in development. Analysis of the developmental expression of mouse AP-2beta has shown that, although some overlap exists, there are distinct patterns of AP-2alpha and AP-2beta 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-2beta 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-2alpha and AP-2gamma were more active in this regard than AP-2beta . In the MRC-5VA cells that express low levels of SOD, both AP-2alpha and AP-2gamma were readily detectable but AP-2beta was not.

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/EBPalpha (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-2alpha variant, AP-2Delta , which lacks the activation domain of AP-2, could still significantly repress Mn-SOD promoter activity. However, another AP-2alpha 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.

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).



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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.


    ACKNOWLEDGEMENTS

We sincerely thank the following individuals for the indicated materials. Lubomir Turek provided the chimeric AP-2-responsive reporter construct and AP-2Delta cDNA. Trevor Williams, Helen Hurst, and Ron Weigel kindly provided expression vectors for AP-2 alpha , beta , and gamma , 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-beta -D-galactopyranoside; kb, kilobase(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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