Linked Common Polymorphisms in the Gelatinase A Promoter Are Associated with Diminished Transcriptional Response to Estrogen and Genetic Fitness*

Sigrid Harendza {ddagger} §, David H. Lovett ¶, Ulf Panzer {ddagger}, Zoltan Lukacs ||, Peter Kühnl ** and Rolf A. K. Stahl {ddagger}

From the Departments of {ddagger}Medicine, Division of Nephrology, ||Department of Pediatrics, and **Transfusion Medicine and Transplantation Immunology, University of Hamburg, Hamburg D-20246, Germany and the Department of Medicine, San Francisco Veterans Affairs Medical Center, University of California, San Francisco, California 94121-1598

Received for publication, November 12, 2002 , and in revised form, March 19, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gelatinase A (matrix metalloproteinase-2) plays a prominent role in multiple biologic processes. Prior studies have established critical roles for gelatinase A transcriptional regulation by defined enhancer elements. To determine possible functional single nucleotide polymorphisms within these elements, we determined the single nucleotide polymorphism distribution within 1,665 bp of the gelatinase A 5'-flanking region, using a healthy homogeneous Caucasian study group of 463 individuals. Among the polymorphisms detected, a G -> A transition at bp –1575 was located immediately 5' to a half-palindromic potential estrogen receptor binding site. In estrogen receptor-positive MCF-7 cells the –1575G allele functioned as an enhancer, whereas the –1575A allele reduced transcription activity significantly. Gel shift assays confirmed that the differences in allelic expression affected binding of the estrogen receptor-{alpha} to this region. Cotransfection experiments with an estrogen receptor-{alpha} expression vector in MDA-MB-231 cells, which do not constitutively express an estrogen receptor, revealed that estrogen receptor is absolutely required for enhancing activity. Allelic distribution analysis indicated that a previously reported C -> T transition within an Sp1 binding site at –1306 was in linkage disequilibrium with the –1575G -> A transition. Luciferase reporter studies of the linked variant –1575A –1306T allele versus the wild type –1575G –1306C allele demonstrated an additive reduction in estrogen-dependent reporter activity. The frequency of the –1575G -> A transition deviated significantly from the expected Hardy-Weinberg distribution in two independently assembled study populations consisting of healthy adult blood donors and newborns of Caucasian origin, both with a calculated 21% reduction in genetic fitness. Gelatinase A is a known estrogen-responsive gene and the demonstration of a loss of function polymorphism within an operational estrogen receptor binding site associated with a decrease in genetic fitness underscores the biologic significance of promoter polymorphism analyses.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The individual components of the relatively large matrix metalloproteinase gene family play multiple roles in the interaction of cells with their surrounding extracellular matrix. As such, this family of evolutionarily conserved enzymes is intimately involved in the diverse physiologic processes that make up normal growth and development, as well as neoplasia, metastasis, angiogenesis, and tissue fibrosis. One member of the matrix metalloproteinase gene family, gelatinase A or matrix metalloproteinase-2, has received considerable attention as a mediator of tumor angiogenesis and metastatic capability (1, 2, 3). In addition to the role of gelatinase A in malignancy, this enzyme has an important function in the normal growth and development of soft tissues. For example, gelatinase A-deficient mice develop normally but have significantly retarded growth rates (4) and diminished corneal and tumor neoangiogenesis (5). One form of the inherited osteolysis (vanishing bone) syndromes was recently found to be the result of a single nucleotide polymorphism (SNP)1 in the coding region of the gelatinase A gene, resulting in a phenotype characterized by an absolute lack of gelatinase A synthesis and severe growth and soft tissue deformities (6).

Although gelatinase A regulatory control has been frequently characterized as "constitutive" in nature, examination of gelatinase A patterns of expression during development or disease states is consistent with multiple levels of cellular control. For example, expression of gelatinase A during nephrogenesis, a classic model of branching morphogenesis, is highly regulated in both a spatial and temporal manner (7). Given these considerations, several groups have attempted to define the transcriptional regulatory elements that provide the cellular- or tissue-specific levels of gelatinase A synthesis. These studies have been performed within both the rat and human genomic contexts and have characterized several motifs operative within defined cellular types (8, 9, 10, 11, 12). For example, Qin et al. (13) demonstrated that a proximal, overlapping binding site for transcription factors AP-2, Sp1, and Sp3 in the human gelatinase A promoter is responsible for high level gelatinase A transcription in glioblastoma cell lines. In contrast, Harendza et al. (8) demonstrated little activity for this region within the context of glomerular mesangial cells and instead defined a potent, tissue-specific enhancer element (denoted RE-1) located between bp –1595 and –1555 relative to the transcriptional start site in the rat gelatinase A gene. A similar element in the human gelatinase A promoter, denoted r2 (bp –1657/–1619) was defined by Frisch and Morisaki (11), and recent studies have demonstrated that this site specifically binds AP-2 and YB-1 complexes (14). The human r2 and rat RE-1 enhancer elements also interact with the p53 protein, resulting in enhanced gelatinase A transcription (12, 15).

Examination of the genetic structure of the promoter regions of other matrix metalloproteinases, including interstitial collagenase and gelatinase B (matrix metalloproteinase-9) have demonstrated specific SNPs that affect either basal promoter activity or the binding of transcription factors, including ets-1 (16, 17, 18). The polymorphism within the gelatinase B promoter affected transcriptional activity and was associated with an increased risk of severe coronary atherosclerosis (17).

Given the central role of gelatinase A in multiple physiologic and disease processes, the complexity of its regulatory control, and the examples provided by the interstitial collagenase and gelatinase B studies, we initiated an examination of the promoter region of the human gelatinase A gene for polymorphisms affecting transcriptional regulation. In particular, we wished to test the hypothesis that polymorphisms in the r2 enhancer region or the proximal overlapping AP-2/Sp1 binding site existed and thereby affected the transcriptional regulation of gelatinase A in growth and development or during disease processes. During the course of this study, Price et al. (19) reported on the distribution of polymorphisms in the human gelatinase A gene and focused on a functional allelic variant at bp –1306 which affected transcription factor Sp1 binding. Although we did not detect polymorphisms within the highly conserved r2 enhancer region, a G -> A transition was mapped to bp –1575 which was immediately adjacent to a potential half-palindromic estrogen receptor binding site. In this report we detail the population distribution of the –1575G/A alleles, demonstrate specific interaction of this region with the estrogen receptor-{alpha} protein, and show that the –1575G -> A transition, which is linked to the –1306C -> T transition, impairs the transcriptional response of gelatinase A to estrogen receptor binding and is associated with a significant decrease in genetic fitness.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Genomic DNA—Blood was collected in EDTA from 463 healthy Caucasian blood donors from Northern Germany and genomic DNA extracted using the QIAamp DNA Minikit (Qiagen). Samples were stored in 30-µl aliquots suspended in 10 mM Tris-HCl and 1 mM EDTA, pH 8.0, and 5-µl aliquots were used as templates for PCR. Heel prick blood from 959 newborns of North German Caucasian origin was collected on standardized filter screening cards and processed as above.

SNP Screening within the 5'-Regulatory Region of the Human Gelatinase A Gene—A set of eight primers was designed following the corrected sequence of Bian and Sun (12; GenBank accession no. AJ298926 [GenBank] ). These priming pairs generated four PCR products spanning the region from bp –1to –1665 relative to the dominant transcriptional start site of the human gelatinase A gene (37). Individual PCR products from the genomic DNA from 20 randomly chosen blood donors were fully sequenced on an ABI 377 sequencer and compared with the original deposited sequence.

Determination of Genotypes—To determine the genotypes for the two SNPs of interest, two genomic DNA fragments of the human gelatinase A 5'-untranslated region were amplified by PCR using the following flanking primer pairs: 5'-CACACCCACCAGACAAGCCT-3' and 5'-TGGGGAATATGGGGAATGTT-3' generates a 349-bp fragment (fragment A) spanning bp –1665 to –1317. The primer pair 5'-CCCAGCACTCTACCTCTTTA-3' and 5'-TGGCAATGTGGGGAGGTTTA-3' generates a 350-bp fragment (fragment B) spanning bp –1316 to –967. When the –1575A allele is present in fragment A, a RcaI restriction site is introduced which is not present in the wild type –1575G allele. When the –1059A allele is present in fragment B, a unique HhaI restriction site is eliminated from the wild type –1059G allele. This permitted restriction enzyme analysis of the PCR products for genotyping of individuals with RcaI for the –1575G/A polymorphism and with HhaI for the –1059G/A polymorphism. Allelic frequency and Hardy-Weinberg distributions were calculated using standard methodologies. To determine the extent of potential linkage disequilibrium between the –1575G/A and the functional Sp1 –1306C/T polymorphism, a randomly chosen subset of 100 samples from the initial pool of 463 subjects was sequenced directly through the –1306 bp region to the –1575 bp region.

Cell Culture—Human breast carcinoma cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection. The cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. For certain transfection experiments cells were grown for a week prior to transfection in phenol red-free RPMI 1640 medium along with 10% fetal calf serum pretreated with dextran-coated charcoal to remove steroid hormones (38).

Human Gelatinase A Promoter Constructs—A set of 5'-deletion constructs of the human gelatinase A 5'-flanking region was prepared by PCR and directionally subcloned into the promoterless luciferase expression vector, pGL2-Basic (Promega), using the KpnI-BglII sites. These constructs were terminated at bp –1665, –1597, –1316, and –1076 relative to the major transcriptional start site and are denoted pT4-Luc1665, pT4-Luc1597, pT4-Luc1316, and pT4-Luc1076, respectively. To test the functional significance of the –1575G/A and –1059G/A polymorphisms, site-specific mutagenesis was performed on wild type genomic templates. These constructs were denominated pT4-Luc1597G1575C1306, pT4-Luc1597A1575C1306, pT4-Luc1076G1059, and pT4-Luc1076A1059. To test the interaction of the –1575G/A polymorphism with the –1306C/T polymorphism, construct pT4-Luc1597A1575C1306 was given the T allele of the –1306 polymorphism by site-specific mutagenesis. This construct was denominated pT4-Luc1597A1575T1306 and compared in transient transfection experiments with construct pT4-Luc1597G1575C1306 containing the wild type C in position –1306.

A second set of luciferase reporter plasmids was constructed by subcloning three concatenated copies of bp –1586 to –1563 into the pGL2-Prom vector (Promega), which includes the heterologous SV40 promoter. The sequences of the forward oligonucleotides used for concatenation are shown with the variant in lowercase; –1575G: 5'-AAGACATAATCgTGACCTCCAATG-3' and –1575A: 5'-AAGACATAATCaTGACCTCCAATG-3'. The constructs were denoted pT4-Luc1575G-P and pT4-Luc1575A-P, respectively. All constructs were sequenced to confirm authenticity.

Transient Transfections—Transient transfections of MCF-7 and MDA-MB-231 cells were performed with polyethyleneimine according to Boussif et al. (39). The respective pT4-Luc expression plasmids and a normalizing pCMV-{beta}-galactosidase plasmid were used in concentrations of 2 µg/well. Total incubation time after transfection was 22 h. All experiments were carried out in quadruplicate and performed independently at least three times. Luciferase and {beta}-galactosidase assays of cell lysates were performed as described (40, 41). Results are expressed as the ratio of luciferase activity to {beta}-galactosidase activity.

For cotransfection experiments, 500 ng of the human estrogen receptor-{alpha} expression plasmid pSG5-HEO (which can be stimulated by estrogen to express the estrogen receptor-{alpha}) or control pSG5 plasmid (the kind gifts of Peter Kushner and Richard Price, University of California, San Francisco) were included in the transfection mixture. Additional cotransfections were performed with equivalent concentrations of the Sp1 expression plasmid, pSG5-Sp1. For these transfections phenol red-free medium and charcoal-treated fetal calf serum were used.

Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts from MCF-7 and MDA-MB-231 cells were prepared as reported previously (8). Synthetic oligonucleotides were annealed and end labeled with polynucleotide kinase and [{gamma}-32P]dATP according to standard methodology. Nuclear extracts were used at 10 µg of protein/reaction in binding buffer with 2 µg of poly(dI-dC) and 1:2 µg of acetylated bovine serum albumin in a total volume of 20 µl. They were incubated for 15 min at 30 °C with the radiolabeled oligonucleotide. The samples were electrophoresed on 4% polyacrylamide and 15% glycerol gels in a buffer containing 1 x Tris borate/EDTA followed by autoradiography. For competition experiments, unlabeled oligonucleotides were added in 50-molar excess to the reaction mixture as described above and incubated for an additional 20 min at room temperature. Antibody depletion experiments were performed by preincubation of the nuclear extracts for 1 h at 4 °C with 1–2 µg/ml of mouse monoclonal anti-human estrogen receptor-{alpha}, rabbit polyclonal anti-human estrogen receptor-{beta}, or control rabbit anti-mouse IgG (Santa Cruz Biotechnology) prior to addition of labeled oligonucleotide and electrophoresis.

Statistical Analysis—Statistical significances were determined for paired comparisons using Student's t test or by analysis of variance for multiple comparisons where appropriate. Statistical levels of significance for deviation from expected Hardy-Weinberg distributions were determined by {chi}2 analysis. The selection coefficient(s) and genetic fitness (w) were calculated using standard methodology.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification and Characterization of SNPs within the 5'-Regulatory Region of the Human Gelatinase A Gene—Using discrete primer pairs encompassing 1665 bp of the 5'-regulatory region of the human gelatinase A gene, PCR was performed with genomic templates from 20 randomly chosen blood donors to assess the nature and extent of nucleotide variation within this region. After sequencing these fragments this study focused on PCR fragments A (spanning bp –1665 to –1317) and B (spanning bp –1316 to –967), each revealing a G -> A transition at position –1575 and at position –1059 (Fig. 1). The –1059G/A polymorphism was used as a control for the population distribution studies because this site is not associated with any defined transcription factor binding site. Sequence analysis indicated that the presence of the –1575A allele introduced an RcaI restriction site into fragment A not present in the wild type allele –1575G. When the –1059A allele is present in fragment B, a unique HhaI restriction site is eliminated.



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FIG. 1.
A, double-stranded DNA sequence between bp –1585 and –1565 of the human gelatinase A 5'-flanking region showing the allelic variant at bp –1575. The allelic variant –1575A can be digested with restriction enzyme RcaI. B, double-stranded DNA sequence between bp –1069 and –1049 with the allelic variant at bp –1059. The variant –1059G can be digested with restriction enzyme HhaI.

 

When PCR fragment A was digested with RcaI, homozygotes with the wild type –1575G allele yielded one undigested 349-bp band on an agarose gel, whereas homozygotes with the –1575A allele yielded two bands, 260 bp and 89 bp in size. Heterozygotes yielded all three bands. Digests of fragment B with HhaI resulted in two bands, 260 bp and 90 bp in size for the wild type –1059G allele and in one undigested band of 350 bp for the –1059A allele (data not shown).

A randomly chosen subset of 100 donors from the original pool was selected and sequenced through the –1575G/A polymorphism and the –1306C/T polymorphism reported by Price et al. (19), to determine whether potential linkage disequilibrium existed between the –1575 and –1306 sites. Only two patterns were detected: –1575G/–1306C and –1575A/–1306T, consistent with complete linkage disequilibrium between the two polymorphism sites.

Survey of the transcription factor data base, Transfac (20), using the sequences surrounding the –1575G/A and –1059G/A alleles indicated that the –1575 bp site is located immediately 5' to a half-palindromic binding site (TGACC) for the estrogen receptor (ERE). This observation prompted a functional analysis of the –1575 region to determine whether the polymorphism adjacent to the half-palindromic ERE affected gelatinase A transcription in response to estrogen.

Allele-specific Binding of Nuclear Proteins to the1575G/A Polymorphic Site—EMSAs were performed to investigate whether the half-palindromic site located between bp –1574 and –1570 was capable of specific interactions with the estrogen receptor and whether this interaction was modified by the –1575G/A transition. Two double-stranded oligonucleotide probes corresponding to the sequence from bp –1586 to –1563 in the gelatinase A promoter, with either G or A at the –1575 polymorphic site, were synthesized, labeled with [{gamma}-32P]dATP, and incubated with nuclear extracts from the estrogen receptor-positive breast carcinoma cell line, MCF-7, or the estrogen receptor-negative breast carcinoma cell line, MDA-MB-231 (Fig. 2). In the presence of the MCF-7 nuclear extract, the radiolabeled 24-bp oligonucleotide containing the wild type G allele showed significant mobility retardation, with formation of a single major oligonucleotide-nuclear protein complex (Fig. 2, lane 2). The intensity of this complex was reduced significantly when MCF-7 nuclear extracts were incubated with the oligonucleotide containing the A allele (Fig. 2, lane 4). No nuclear protein-DNA binding was observed when the respective oligonucleotides were incubated with nuclear extracts from the estrogen receptor-negative MDA-MB-231 cells (Fig. 2, lanes 6 and 8).



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FIG. 2.
EMSA with radiolabeled 24-bp oligonucleotides containing either the –1575G or –1575A allele and nuclear extracts from estrogen receptor-positive MCF-7 cells or estrogen receptor-negative MDA-MB-231 cells. The mobilities of the labeled oligonucleotides without added nuclear extracts are shown in lanes 1, 3, 5, and 7. The arrow localizes a major oligonucleotide-nuclear protein complex in lane 2 using the –1575G allele, which is reduced significantly with the –1575A allele oligonucleotide. No oligonucleotide-nuclear protein complex was detected using extracts from MDA-MB-231 cells.

 

The specificity of MCF-7 nuclear protein binding to the –1586/–1563 oligonucleotide was confirmed by competition experiments (Fig. 3). A 50-molar excess of cold –1575G oligonucleotide strongly competed for MCF-7 nuclear protein binding, resulting in the disappearance of the major shifted complex (Fig. 3, lane 3), whereas a 50-molar excess of cold –1575A oligonucleotide revealed only partial competition (Fig. 3, lane 4). No competition was observed when a cold oligonucleotide composed of the scrambled –1586/–1563 sequence was used (Fig. 3, lane 5), demonstrating the sequence specificity of MCF-7 nuclear protein binding.



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FIG. 3.
EMSA with the radiolabeled 24-bp oligonucleotide containing the –1575G allele and nuclear extracts from MCF-7 cells. Specificity of nuclear protein binding is demonstrated by cold competition with 50-molar excess of oligonucleotide with the –1575G allele (I.) versus the –1575A allele (II.) or a scrambled sequence (III.).

 

The formation of the oligonucleotide-nuclear protein complex was reduced significantly when the MCF-7 nuclear extracts were preincubated with a specific mouse monoclonal anti-human estrogen receptor-{alpha} antibody (Fig. 4, lane 3). Preincubation with a polyclonal rabbit anti-human estrogen receptor-{beta} antibody (Fig. 4, lane 4) or control rabbit anti-mouse IgG (Fig. 4, lane 5) had no significant effect on the formation of the oligonucleotide-nuclear protein complex. These EMSA studies confirm a sequence-specific interaction of the human estrogen receptor-{alpha} protein present in MCF-7 nuclear extracts with the half-palindromic sequence. The absence of estrogen receptor-{beta} protein binding to this site is consistent with the fact that MCF-7 cells do not express this form of the estrogen receptor. The competition experiments are consistent with estrogen receptor-{alpha}/half-palindromic sequence interactions, which have a binding affinity of at least 100-fold less for the estrogen receptor protein compared with a full palindromic sequence (21).



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FIG. 4.
EMSA with the radiolabeled 24-bp oligonucleotide containing the –1575G allele and MCF-7 nuclear extracts: incubation with anti-estrogen receptor antibodies. Antibody depletion analysis was performed by preincubating the nuclear extracts with anti-estrogen receptor-{alpha} antibody (lane 3), anti-estrogen receptor-{beta} antibody (lane 4), or anti-mouse IgG as a negative control (lane 5).

 

Functional Characterization of the Half-palindromic ERE on Gelatinase A Transcription—A series of deletion constructs of the gelatinase A 5'-flanking region was prepared and sequenced to confirm that they contained exclusively the wild type alleles reported by Price et al. (19). Estrogen receptor-positive MCF-7 cells were used for transient transfection reporter studies and the results standardized by cotransfection with a {beta}-galactosidase expression vector. The luciferase values obtained with transient transfection with the construct, pT4-Luc1665 were assigned a relative activity of 100% (Fig. 5), to permit comparison with the patterns of reporter activity obtained by Qin et al. (13), using the same type of constructs. Low levels of luciferase activity were seen with construct pT4-Luc1076 (Fig. 5), and a modest increase in activity was seen with the longer construct, pT4-Luc1316, which includes the Sp1 binding site at –1306. A 2-fold increase in reporter activity was obtained with construct pT4-Luc1597, which includes the estrogen receptor half-palindromic binding site, whereas the longest construct, pT4-Luc1665, revealed a similar pattern of silencing activity as described by Bian et al. (12). These studies are consistent with the presence of an enhancer activity within the 281-bp segment extending from bp –1316 to –1586, which includes the estrogen receptor half-palindromic binding sequence –1575G/A TGACC.



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FIG. 5.
Transient transfection of MCF-7 cells with deletion constructs of the 5'-flanking region of the human gelatinase A promoter driving luciferase as a reporter gene. The known cis-acting elements, p53 and Sp-1, and the putative half-palindromic estrogen receptor binding sequence are denoted. Data are presented as the ratios of luciferase (LUC) versus {beta}-galactosidase activities with the construct pT4-Luc1665 assigned a value of 1. Results are the means of three independent transfection experiments.

 

To assess directly the functional significance of the –1575G/A polymorphism on gelatinase A transcriptional activity, the wild type pT4-Luc1597G1575C1306 construct was modified by site-specific mutagenesis to generate construct pT4-Luc1597A1575C1306 (Fig. 6A). Transient transfection of MCF-7 cells revealed that the pT4-Luc1597A1575C1306 construct exhibited less than 50% of the luciferase reporter activity of the wild type pT4-Luc1597G1575C1306 construct (Fig. 6B). When the same constructs were transfected into estrogen receptor-negative MDA-MB-231 cells, no differences in luciferase activities were detected. For the second polymorphism at bp –1059 a similar set of constructs, denoted pT4-Luc1076G1059 and pT4-Luc1076A1059, was prepared. When these constructs were transfected into MCF-7 and MDA-MB-231 cells no significant differences in luciferase activity were observed (not shown).



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FIG. 6.
A, luciferase reporter constructs containing 1597 bp of the 5[prime]-flanking region of the human gelatinase A promoter as either the –1575G or –1575A alleles in context with the –1306 wild type allele. B, transient transfection of MCF-7 and MDA-MB-231 cells with the G or A allelic constructs. The construct containing the G allele was assigned a value of 1. Results are the means of at least three independent transfection experiments (* p < 0.05).

 

For further investigation of the functional significance of the polymorphism within the half-palindromic estrogen receptor binding sequence, two constructs with either G or A were prepared consisting of three concatenated copies of the 24-bp sequence flanking the –1575 polymorphism cloned in front of a heterologous SV40 promoter (Fig. 7A). Transient transfection of MCF-7 cells with the wild type pT4-Luc1575G-P construct yielded a large increase in luciferase reporter activity compared with the control SV40 vector pGL2-Prom alone (Fig. 7B). In contrast, the construct pT4-Luc1575A-P yielded significantly less luciferase reporter activity (<50%). Transfection of the pT4-Luc1575G-P and pT4-Luc1575A-P constructs into the estrogen receptor-negative MDA-MB-231 cells generated only minimal luciferase reporter activity, consistent with an estrogen receptor requirement for the enhancer activity of the –1586/–1563 segment.



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FIG. 7.
A, construct design for comparison of allelic variance on enhancer activity of a 24-bp region centered on the –1575 site. Each construct includes three concatenated copies of the 24-bp sequence cloned into a vector containing the heterologous SV40 promoter. B, allelic expression was measured as the -fold increase in luciferase activity relative to the activity of the pGL2-Prom vector, assigned a value of 1.

 

An additional series of experiments used transfection of estrogen receptor-negative MDA-MB-231 cells with the estrogen-responsive estrogen receptor-{alpha} expression plasmid pSG5-HEO and the pT4-Luc1597G1575C1306, pT4-Luc1597A1575C1306, or the pT4-Luc1597A1575T1306 reporter constructs. The results of these studies are summarized in Fig. 8. Reporter activities of MDA-MB-231 cells transfected with the reporter constructs and the control expression vector pSG5 were assigned a relative luciferase activity of 1. Cotransfection of the estrogen receptor-{alpha} expression plasmid pSG5-HEO in the absence of estradiol did not significantly affect luciferase activity in any of the constructs, as expected. In contrast, the inclusion of 10 nM estradiol induced an approximate 3.5-fold increase in reporter activity by the wild type pT4-Luc1597G1575C1306 construct. The estrogen responsiveness of the pT4-Luc1597A1575C1306 and the pT4-Luc1597A1575T1306 reporter constructs was only half of that of the wild type construct. The partial loss in luciferase reporter activities of the pT4-Luc1597A1575C1306 and the pT4-Luc1597A1575T1306 plasmids is consistent with the partial loss in estrogen receptor protein binding (as determined by EMSA) introduced by the –1575G -> A transition. This experiment confirms the specificity of estrogen receptor-{alpha} interaction with the half-palindromic binding site and the functional significance of the –1575G -> A transition. In addition, the functional Sp1 binding site C -> T transition does not directly affect the gelatinase A promoter response to estradiol.



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FIG. 8.
Luciferase expression of constructs pT4-Luc1597G1575C1306, pT4-Luc1597A1575C1306, and pT4-Luc1597A1575T1306 in MDA-MB-231 cells with cotransfection of a control pSG5 expression plasmid, pSG5-HEO (expressing the human estrogen receptor-{alpha}), and pSG5-HEO plus 10 nM estradiol. Transfections were performed in phenol red-free RPMI 1640 medium supplemented with dextran-coated charcoal treated fetal calf serum. Data are given as ratios of luciferase versus {beta}-galactosidase activities with the pSG5-vector assigned a value of 1.

 

Estrogen receptor protein and Sp1 interactions have been defined for several promoters and include direct physical interactions of the two proteins, synergistic activation of promoter at adjacent binding sites or simple additive effects of physically discrete binding elements (22, 23, 24, 25, 26). A final set of experiments was designed to explore whether any of these discrete models of ERE and Sp1 interaction applied to the gelatinase A promoter within the context of the –1575 and –1306 polymorphisms. The results of these studies are summarized in Fig. 9. Cotransfection of the Sp1 expression vector pSG5-Sp1 with the wild type pT4-Luc1597G1575C1306 construct yielded an approximate 5-fold increase in reporter activity, and, as shown in Fig. 8, cotransfection with the pSG5-HEO in the presence of estradiol induced an approximate 4-fold increase in activity. Transfections that employed both the Sp1 expression plasmid and the pSG5-HEO plasmid in the presence of estradiol demonstrated additive increases in luciferase activity of ~10–12-fold. These responses were each decreased by ~50% when the pT4-Luc1597A1575T1306 reporter construct was used. Thus, each discrete transition at –1575 and –1306 reduced the response to either the estrogen receptor or Sp1 proteins by half, respectively, and the total amount of reporter activity reflects the sum of these responses. This pattern is most consistent with the ERE-Sp1 interaction model described by Martini et al. (26), in which physically separated half-palindromic ERE and Sp1 binding sites regulate transcriptional responses in a simple, additive manner which does not involve direct ERE-Sp1 protein interactions.



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FIG. 9.
Wild type construct pT4-Luc1597G1575C1306 and double mutant construct pT4-Luc 1597A1575T1306 expressed in MDA-MB-231 cells, cotransfected with the control pSG5 expression plasmid, the Sp1 expression plasmid pSG5-Sp1, as well as the estrogen receptor-{alpha} expression plasmid pSG5-HEO with and without stimulation of 10 nM estradiol, and with and without the addition of pSG5-Sp1. The RPMI 1640 medium for these transfections was phenol red-free and supplemented with dextran-coated charcoal-treated fetal calf serum. Expression was measured as the -fold increase in luciferase activity relative to the activity of the pSG5 expression plasmid in context with the wild type construct pT4-Luc1597G1575C1306, assigned a value of 1.

 

Restriction analysis was used for determination of the distribution of genotypes at bp –1575 and –1059 within a group of 463 healthy Caucasian blood donors (Table I). Although the genotype distribution for the –1059G/A polymorphism was in Hardy-Weinberg equilibrium, (predicted values were GG:GA: AA = 79.2%:19.6%:1.2%; actual enumerated values were GG: GA:AA = 79.5%:18.8%:1.7%), the genotype distribution for the –1575G/A polymorphism within the total study population was not. The values predicted by the assumption of Hardy-Weinberg equilibrium for the polymorphism at bp –1575 were GG:GA:AA = 59.1%:35.5%:5.3%, respectively, whereas the actual enumerated values were GG:GA:AA = 56.4%:41.0%:2.6%, respectively (p < 0.05 by {chi}2 analysis). Calculation of genetic fitness (w) revealed a value of 0.79, or a selection coefficient s (where w = 1-s) of 0.21, with a p value of less than 0.05.


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TABLE I
Genotype and allelic frequencies of the1575G/A and1059G/A polymorphisms in the 5'-flanking region of the human gelatinase A promoter

 

To confirm the deviation of the –1575G/A transition from apparent Hardy-Weinberg equilibrium and to gain potential insights into possible sources of selection pressure, a second, independently constituted study group of 959 newborns of North German Caucasian background (471 males, 488 females) was assessed. As summarized in Table II, the genotype frequencies for the total newborn study group and for the male/female subgroups were virtually the same as observed with the healthy blood donor group. The data also revealed equivalent degrees of deviation from the expected Hardy-Weinberg genotype frequencies values. The predicted values for equilibrium were GG:GA:AA = 58.9:35.6:5.5, respectively, whereas the enumerated values were GG:GA:AA = 56.1:41.4:2.5, respectively (p < 0.05 by {chi}2 analysis). Equivalent degrees of deviation from equilibrium were present in both the male and female subgroups.


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TABLE II
Genotype and allelic frequencies of the1575G/A polymorphism in an independently derived study population of 959 Northern German Caucasian newborns

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gelatinase A is an important component of multiple physiologic and pathophysiologic processes, including cell proliferation, angiogenesis and tumor metastasis (27, 28). Prior studies from this and other laboratories have defined several critical regulatory elements in the 5'-flanking regions of the human and rat gelatinase A genes that regulate spatial and temporal patterns of expression during growth and development and following defined forms of tissue injury (8, 9, 12, 13, 29). In this study we screened a normal Caucasian population (n = 463) from Northern Germany for SNPs within the immediate 5'-flanking region of the human gelatinase A gene with the intent to define potential allelic variants within the previously reported sites of transcription factor binding. During the course of this study Price et al. (19) reported on the SNP distribution within the entire gelatinase A gene from 32 Caucasians, including ~2 kb of the 5'-flanking region. Within this region six polymorphisms were defined in this sample population, including the –1575G/A transition analyzed in this study. In our larger study population the six SNPs reported by Price et al. (19) were detected, and an additional –1059G/A transition was detected with an allelic frequency of 0.111 (Table I). Interestingly, Martignetti et al. (6) reported additional polymorphisms not described by Price et al. (19) in the gelatinase A gene in a sample population of three families with the inherited osteolysis syndrome and absent gelatinase A synthesis. In this sample drawn from Saudi Arabia, additional polymorphisms at Y244X and R101H were described, underscoring the contribution of ethnic or racial backgrounds for these types of analyses.

Gelatinase A is an estrogen-responsive gene and is up-regulated in cultured vascular smooth muscle cells or cultured glomerular mesangial cells after exposure to estradiol (30, 31). Estradiol also stimulates gelatinase A synthesis by granulosalutein cells (32). Estrogen replacement reduces age-associated remodeling in rat mesenteric arteries, an event correlated with induction of vascular wall gelatinase A synthesis (33). Expression of gelatinase A is enhanced in breast remodeling stimulated by estradiol, a process associated with increased cellular motility (34).

Although our analysis of 463 North German Caucasian blood donors did not reveal detectable polymorphisms within the previously defined enhancer elements or other reported transcription factor binding sites, examination of the sequence immediately 3' to the –1575G/A transition revealed an incomplete, or half-palindromic binding site (–1575G/A TGACC) for the estrogen receptor.

As detailed under "Results," further analysis using a combination of EMSA, specific estrogen receptor antibodies, and functional luciferase reporter assays provides evidence for the functional activity of this half-palindrome site and shows that the G -> A transition at bp –1575 significantly reduces estrogen receptor-{alpha} binding and gelatinase A transactivation. Notably, the estrogen receptor-negative breast carcinoma cell line MDA-MB-231 did not demonstrate estrogen receptor protein binding or enhancement in luciferase activities, but responded to cotransfection with the estrogen receptor-{alpha} expression plasmid and exposure to estradiol. This observation is consistent with the data presented by Price et al. (19), who reported that the –1575G/A polymorphism was not functional when tested within the background of other estrogen receptor-negative cells, including the HEK293 (embryonic kidney), RAW264.7 (macrophage), and A10 (tubular epithelium) cell lines.

The consensus estrogen response element sequence is 5'-GGTCANNNTGACC-3', where the N represents the relative location of the –1575G/A polymorphism and is thus not precisely located within the DNA sequence that directly contacts the estrogen receptor protein. Kato et al. (21) defined the estrogen receptor binding characteristics of half-palindromic sequences and observed that the binding affinity of such sequences is at least 100-fold less than for a complete palindromic sequence. Given the relatively low affinity of the estrogen receptor protein for half-palindromic sites, Kato et al. (21) suggested that the composition of the immediately adjacent nucleotides, such as the –1575 site, may directly influence receptor protein binding or potential partner protein interactions required for binding. We note that the –1575G -> A transition is associated with a diminution in estrogen receptor protein binding and transactivation activity, and the model outlined by Kato et al. (21) is consistent with our experimental observations.

The –1575G -> A transition was found to be linked to a –1306C -> T transition shown previously to diminish Sp1 protein binding and transactivation (19). Luciferase constructs including both transitions revealed arithmetically decreased responses to estrogen receptor and Sp1 expression plasmid transfection, suggesting that the Sp1 and estrogen receptor proteins do not physically interact on the gelatinase A promoter, but rather act independently in a simple additive fashion.

Given the sample sizes used in this study we were able to determine directly genotype and allelic frequencies by observation as opposed to estimation. The nonfunctional –1059G/A allele was found to be distributed within the confines of the Hardy-Weinberg equilibrium, whereas the functional –1575G/A allele distribution revealed a statistically significant deviation (2.6% AA observed versus 5.3% AA expected) from the Hardy-Weinberg equilibrium with a corresponding reduction in genetic fitness of 21%. The mean ages of the male and female blood donors were 39 and 38 years, respectively, and given the requirement for general good health in this population, the selection against the AA allele is not likely because of common adult diseases associated with increased mortality. Given the homogeneous nature of the study population with no issues of admixture, it is more reasonable to postulate that the deviation from the expected distribution is related to decreased reproductive or prenatal viability in the AA genotype. This hypothesis is strongly supported by our analysis of a second independently constituted study population of 959 newborns of North German Caucasian origin, in which virtually identical degrees of deviation from the expected Hardy-Weinberg values were observed, in both male and female subjects. Finally, statistically identical genotype frequencies for the –1575G/A allele were observed in a third, independently constituted study of 628 patients with idiopathic membranous glomerulonephritis (data not shown). The median age in this third study group was 62 years, again supporting the hypothesis that selection pressure is directed to reproduction or prenatal viability. It is unlikely that these genotype frequency results are the consequence of genotyping errors because the data obtained by restriction enzyme analysis were fully corroborated in a subset of 100 randomly chosen subjects analyzed by direct sequencing.

Gelatinase A plays an important role for estrogen-regulated trophoblast and stromal remodeling during implantation (35). Although there are no extant data on the reproductive efficiency of the gelatinase A-deficient mouse, deficiency of the closely related gelatinase B results in significantly impaired reproduction efficiency (36). Because the –1575G/A transition results in an incomplete loss of estrogen responsiveness, we cannot rule out that the –1575A variant may be in linkage disequilibrium with other regulatory regions of the gelatinase A promoter that affect transcriptional regulation or the response to estrogen. This question is currently under investigation.

In summary, we have demonstrated that the human gelatinase A promoter includes a functional polymorphism at bp –1575 which affects the transcriptional response to estrogen and genetic fitness. Future studies, including more extensive haplotype analyses of the gelatinase A gene, may be expected to provide additional insights into the regulatory control of this critical gene.


    FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant HA 2056/3–3 (to S. H.) and National Institutes of Health Grant DK 39776 (to D. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Universitätsklinikum Hamburg-Eppendorf, Zentrum für Innere Medizin, Medizinische Klinik IV, Nephrologie und Osteologie, Pavillon N26, Martinistrasse 52, Hamburg D-20246, Germany.

1 The abbreviations used are: SNP, single nucleotide polymorphism; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift analysis; ERE, estrogen response element. Back



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 RESULTS
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