HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 8, 6296-6302, February 22, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/8/6296    most recent M107865200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fujimoto, T. Articles by Strauss, J. F. Search for Related Content PubMed PubMed Citation Articles by Fujimoto, T. Articles by Strauss, J. F., III

A Single Nucleotide Polymorphism in the Matrix Metalloproteinase-1 (MMP-1) Promoter Influences Amnion Cell MMP-1 Expression and Risk for Preterm Premature Rupture of the Fetal Membranes^*

Toshio Fujimoto, Samuel Parry, Margrit Urbanek^§, Mary Sammel^¶, George Macones, Helena Kuivaniemi, Roberto Romero, and Jerome F. Strauss III^**

From the  Center for Research on Reproduction and Women's Health, the ^§ Department of Genetics, and the ^¶ Department of Biostatistics and Clinical Epidemiology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, and the  Perinatology Research Branch, NICHHD, National Institutes of Health, Hutzel Hospital, Detroit, Michigan 48201

Received for publication, August 15, 2001, and in revised form, November 27, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interstitial collagen gives fetal membranes tensile strength, and membrane rupture has been attributed to collagen degradation. A polymorphism at 1607 in the matrix metalloproteinase-1 (MMP-1) promoter (an insertion of a guanine (G)) creates a core Ets binding site and increases promoter activity. We investigated whether this polymorphism is functionally significant for MMP-1 expression in amnion cells and whether it is associated with preterm premature rupture of the membranes (PPROM). The 2G promoter had >2-fold greater activity than the 1G allele in amnion mesenchymal cells and WISH amnion cells. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and PPROM. We conclude that the 2G allele has stronger promoter activity in amnion cells, that it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and that this polymorphism contributes to the risk of PPROM.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Complications resulting from idiopathic preterm birth, defined as delivery before 37 weeks of gestation, account for the majority of perinatal deaths of infants without anomalies in developed nations (1, 2). Preterm premature rupture of the membranes (PPROM)¹ occurs in ~1% of all pregnancies and is associated with 30-40% of preterm deliveries (3). PPROM is thus the leading identifiable cause of preterm birth and its complications, including respiratory distress syndrome, neonatal infection, and intraventricular hemorrhage (3, 4). Consequently, identification of markers that identify patients at risk for PPROM is a subject of considerable interest.

Interstitial collagens (type I and type III collagen) of the amnion confer upon the fetal membranes tensile strength (5). Rupture of fetal membranes at term as well as preterm has been attributed, in part, to degradation of the collagens in the extracellular matrix. The breakdown of the interstitial collagens is mediated by matrix metalloproteinases (MMPs). The first step in interstitial collagen catabolism is catalyzed by collagenases including matrix metalloproteinase 1 (MMP-1), the primary collagenase elaborated by fibroblasts (6). It is also produced by monocytic cells (7). MMP-1 is produced as a proenzyme; after proteolytic activation, it cleaves through the fibrillar triple helix at a specific site, producing fragments that can be further degraded by other MMPs, including the gelatinases MMP-2 and MMP-9 (8-12). Evidence that MMP activity is important for fetal membrane rupture includes the observation that fetal membrane MMP expression increases at the time of parturition (13, 14). Moreover, MMP levels are elevated in human amniotic fluid in association with PPROM (15-18).

A single nucleotide polymorphism at nucleotide 1607 in the MMP-1 promoter in which insertion of a guanine nucleotide (G) creates a core binding site for Ets transcription factors (5'-GGAT-3') has been reported to increase MMP-1 promoter activity (19). In general, Ets transcription factors cooperate with activator protein-1 (AP-1) to enhance the activity of several MMP promoters. However, this polymorphism has yet to be shown to influence transcription of the endogenous MMP-1 gene and to affect production of MMP-1 protein. Because MMP-1 is the primary collagenase involved in interstitial collagen degradation by mesenchymal cells, and its concentrations in amniotic fluid are increased in association with PPROM (18), we investigated whether this insertion/deletion polymorphism in the MMP-1 promoter is functionally significant for MMP-1 expression in amnion cells and whether the polymorphism is associated with PPROM. We show for the first time that the 1607 polymorphism indeed contributes to the regulation of endogenous MMP-1 gene transcription, the level of enzyme produced by mesenchymal cells, and is associated with risk of an obstetrical complication caused by increased catabolism of interstitial collagen.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Subjects

Subjects in this study were offspring of African American women receiving obstetrical care at the Hospital of the University of Pennsylvania, Philadelphia, and Hutzel Hospital, Detroit. The study was approved by the respective Institutional Review Boards, and informed consent was obtained from mothers prior to collection of the samples. Controls were derived from singleton pregnancies delivered at term of mothers with no prior history of PPROM or preterm birth. Cases were singleton pregnancies complicated by PPROM, defined as fetal membrane rupture prior to 37 weeks of completed gestation. PPROM was diagnosed by vaginal pooling of fluid and positive nitrazine and fern tests. Pregnancies with fetal malformations or medical complications requiring induction of labor were excluded from both the control and case groups.

Isolation and Culture of Amnion Mesenchymal Cells

Cultures of amnion mesenchymal cells were prepared from fetal membranes obtained from patients with term gestations. After separation of the amnion from the chorion, pieces of amnion were minced and then digested with 1 mg/ml collagenase A (Roche Molecular Biochemicals) in 1× Hanks' balanced salt solution (Invitrogen) at 37 °C with gentle shaking for 2 h (20). After digestion, the suspension was filtered through a 0.28-mm wire mesh, and cells were pelleted by centrifugation at 3,000 rpm for 10 min. The cells were resuspended in 1× Hanks' balanced salt solution and then centrifuged. The cell pellet was resuspended in 3 ml of 1× Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) and layered onto a discontinuous Percoll gradient (5/20/40/60%, v/v). The gradient was centrifuged at 1,500 rpm for 20 min. The cells were collected from the interface of the 20-40% layer, washed by centrifugation in DMEM, and then plated in medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen). Cells were collected after 7 days of culture and frozen for subsequent use after one passage.

Immunocytochemistry

Cells cultured after a single passage were fixed with paraformaldehyde (4%) and stained for vimentin (Dako Corporation, Carpinteria, CA) and cytokeratin-18 (Sigma) using Vectastain ABC kit reagents and mouse IgG (Vector Laboratories, Burlingame, CA).

Cell Culture and Transfection

A2058 melanoma cells were cultured in DMEM, 10% FBS, and 0.1% gentamicin. WISH amnion-derived cells were cultured in Kennett's HY medium (Invitrogen), containing 1× OPI (0.15 mg/ml oxalacetic acid, 50 µg/ml sodium pyruvate, 0.2 unit/ml insulin), 2% L-glutamine, 10 mM HEPES, 2% FBS, and 0.1% gentamicin. Amnion mesenchymal cells were cultured in DMEM, 10% FBS, and 1× Antibiotic-Antimycotic (Invitrogen). Human cervical smooth muscle cells isolated from uteri of nonpregnant women removed for benign disease were purchased from Clonetics (San Diego). The human cervical smooth muscle cells were grown in smooth muscle cell growth medium-2 supplemented with 5% FBS (Clonetics). This medium contains 0.5 ng/ml human epidermal growth factor, 1.0 ng/ml human fibroblast growth factor, 5 µg/ml insulin, 50 µg/ml gentamicin, and 50 µg/ml amphotericin B. All cells were maintained at 37 °C under an atmosphere of 5% CO₂ in air.

For transfection, 1 × 10⁵ cells were seeded in each well of a 12-well plastic culture plate. Cells were transfected using FuGENE^TM 6 Transfection Reagent (Roche Molecular Biochemicals) with 0.5 µg of the pGL3 vector containing a 4,372 bp of MMP-1 promoter fragment coupled to firefly luciferase reporter gene. The promoter fragment, which has been described previously, was a generous gift from Drs. Joni L. Rutter and Constance E. Brinckerhoff (Dartmouth Medical School). The sequences of the constructed reporter plasmids containing either the 1G or 2G at the 1607 polymorphism were confirmed by DNA sequence analysis. In each transfection 0.5 µg of pCMV--galactosidase plasmid (Promega, Madison, WI) was included to correct for transfection efficiency. During the transfection, cells were cultured in medium supplemented with 1% FBS for 24 h.

Luciferase and -Galactosidase Assays

After 24 h of culture, transfected cells were collected into lysis buffer, and luciferase and -galactosidase assays were performed using reagent systems purchased from Promega. Relative luciferase units were calculated as the ratio of luciferase light units to -galactosidase activity.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays

Amnion mesenchymal cells were incubated with 2% dimethyl sulfoxide (Me₂SO) or 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 h, and the nuclear proteins were extracted as described previously (21). The following double-stranded oligonucleotide probes were constructed (19): 1G sense, 5'-AAATAATTAGAAAGATATGACTTATCTCAAATCAA-3'; 2G sense, 5'-AAATAATTAGAAAGGATATGACTTATCTCAAATCAA-3'; 78/59, AP-1 sense, 5'-AAAGCATGAGTCAGACACCT-3'; an oligonucleotide containing a consensus Ets binding site (indicated in bold), 5'-GGGCTGCTTGAGGAAGTATAAGAAT-3'; and an oligonucleotide with a mutated consensus Ets binding site (bold), 5'-GGGCTGGAGAGAGTATAAGAAT-3'. The double-stranded synthetic oligonucleotides were labeled with T4 polynucleotide kinase (Invitrogen) and [³²P]ATP. The electrophoretic mobility shift assay binding reaction was carried out in 10 mM Tris-HCl (pH 7.8), 100 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl₂, 4% glycerol. Briefly, 15 µg of nuclear protein, 2 × 10⁵ cpm of ³²P-labeled double-stranded oligonucleotide probe (2 ng), 4 µg of poly(dI·dC) (Roche Molecular Biochemicals) with or without unlabeled competitor probe were incubated in a total volume of 30 µl. Reaction mixtures were incubated at room temperature for 30 min and then subjected to 8% PAGE at 200 V for 2 h. The dried gels were then exposed to x-ray film.

Northern Analysis

Cultures of amnion mesenchymal cells were incubated with 2% Me₂SO vehicle or 50 ng/ml PMA in medium supplemented with 1% FBS for 24 h. Total RNA was extracted from the cultures with TRIzoL reagent (Invitrogen) according to the manufacturer's instructions. Ten µg of total RNA was separated on 1% agarose-formaldehyde denaturing gels and transferred to nylon membranes (Amersham Biosciences, Inc.). Membranes were probed with a radioactively labeled MMP-1 cDNA fragment (22) and subsequently a cDNA encoding human 28 S rRNA.

Quantitative Real Time Reverse Transcription PCR

Amnion mesenchymal cells were incubated with 2% Me₂SO vehicle or 50 ng/ml PMA for 24 h. Total RNA was extracted from 15 cell cultures with different genotypes with respect to the nucleotide 1607 MMP-1 promoter polymorphism (four 1G/1G homozygotes; eight 1G/2G heterozygotes, and three 2G/2G homozygotes) using TRIzoL reagent. Five µg of total RNA was reverse transcribed to single-stranded cDNA using 25 ng/µl oligo(dT)₁₅ primer (Promega), 1 unit/µl RNase inhibitor from human placenta (Roche Molecular Biochemicals), and 10 units/µl Moloney murine leukemia virus reverse transcriptase (Promega) as described by the manufacturer.

Quantitative real-time PCR was performed to assess the induction of MMP-1 mRNA in mesenchymal cells with different 1607 MMP-1 promoter genotypes in response to 50 ng/ml PMA. Primers for the analysis of the human MMP-1 mRNA were designed with the Primer Express software package that accompanies the Applied Biosystems 7700 sequence detector (PerkinElmer Life Sciences). The forward and reverse primers were designed to span the fourth intron: forward, 5'-AGATGAAAGGTGGACCAACAATTT-3'; and reverse, 5'-CCAAGAGAATGGCCGAGTTC-3'. The real-time PCR used a 90 nM concentration of each primer and 12.5 µl of SYBR Green PCR Master Mix (Applied Biosystems). Analysis of the PCR products revealed the presence of a single 78-bp PCR product. To account for differences in starting material, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was quantified using primers and probe from Applied Biosystems as described by the manufacturer. The target and GAPDH PCRs were performed in separate tubes in triplicate, and the average threshold cycle for the triplicates was used in all subsequent calculations.

Quantitative Real Time PCR for Nascent Transcripts

To assess transcription of the MMP-1 gene we quantified MMP-1 nascent transcripts using primers that detected intronic sequences. To limit the possibility of detection of genomic DNA, 5 µg of total RNA was treated with RQ1 RNase-free DNase (Promega) for 30 min at 37 °C before reverse transcription with Moloney murine leukemia virus. For the human MMP-1 and human GAPDH nascent transcripts, reverse primers were designed in the eighth and third introns, respectively: MMP-1, 5'-GCCCTAACATTCTCTGCACT-3'; GAPDH, 5'-TAGTTGCCTCCCCAAAGCAC-3'. The resulting cDNA was diluted 10-fold in sterile water, and aliquots were subjected to quantitative real time PCR. The forward and reverse primers for MMP-1 were designed in eighth exon: forward, 5'-ACGGATACCCCAAGGACATCT-3'; reverse; 5'-CTCAGAAAGAGCAGCATCGATATG-3'. For the GAPDH nascent transcripts, primers were designed in the third exon: forward, 5'-GATTCCACCCATGGCAAATT-3'; reverse, 5'-AAGATGGTGATGGGATTTCCATT-3'. Optimization of the PCR indicated that a 90 nM concentration of each primer should be used in each 25-µl reaction to eliminate primer dimer formation. Analysis of PCR products indicated the presence of a single product for both MMP-1 (77 bp) and GAPDH (83 bp).

MMP-1 Protein Analysis

Amnion mesenchymal cells (1 × 10⁵ cells/well) were seeded into six-well plastic culture plates and cultured in DMEM supplemented with 10% FBS to 90% confluence. Cells were then incubated with 2% Me₂SO vehicle or 50 ng/ml PMA in DMEM supplemented with 1% FBS for 24 h. Conditioned medium was then collected and analyzed for MMP-1 by Western blotting (22) and ELISA (Oncogene Research Products, Boston) as described by the manufacturer. The ELISA detects both pro-MMP-1 and activated MMP-1.

DNA Samples

DNA was extracted from cultured cells and umbilical cords, cheek swabs, or umbilical blood samples from African American newborns from singleton pregnancies complicated by PPROM and newborns from singleton pregnancies delivered at term. DNA from cultured cells and from umbilical cords was isolated by digestion with proteinase K and extraction with phenol/chloroform/isoamyl alcohol (23). DNA from cheek swabs was isolated in 50 mM NaOH and 1 M Tris (pH 8.0). DNA from blood samples was extracted using QIAamp^® DNA Mini Kits (Qiagen, Valencia, CA) as described by the manufacturer. The extracted genomic DNA was stored at 70 °C for subsequent use.

MMP-1 Promoter Genotyping

We employed two different methods to genotype the nucleotide 1607 MMP-1 promoter polymorphism.

Method 1 (Genescan/Genotyper)-- Primers that flank the insertion/deletion polymorphism in the MMP-1 promoter used for PCR were: forward primer, bp 1650 to 1631, 5'- FAM-GTTATGCCACTTAGATGAGG; and reverse primer, bp 1522 to 1503, 5'-TTCCTCCCCTTATGGATTCC-3'. PCR was performed in a 50-µl reaction volume containing 100 ng of genomic DNA, 25 pmol of each primer, 0.2 mM dNTPs, 1 × reaction buffer, and 2.5 units of Pfu DNA polymerase (Stratagene). The PCRs were performed in a 9600 Gene Amp PCR thermal cycler (PerkinElmer Life Sciences) in 35 cycles (5 min at 94 °C, 1 min at 94 °C, 1 min at 55 °C, 1 min at 68 °C, followed by a 7-min extension at 72 °C). PCR products were electrophoresed in the presence of an internal size standard (Genescan 500) on 4% acrylamide, 5 M urea denaturing gels using a 377 DNA sequencer (PE Applied Biosystems). Gels were run for 2.5 h at 3,000 V. Genotypes were determined using the Genescan Analysis and Genotyper programs (PE Applied Biosystems).

Method 2 (Restriction Endonuclease XmnI Digestion)-- The sequences of PCR primers were: forward primer, bp 1695 to 1675, 5'-TGCTGAGAATGTCTTCCCATT-3'; and reverse primer, bp 1578 to 1606, 5'-TCTTGGATTGATTTGAGATAAGTGAAATC-3'. Two mismatches were introduced in the reverse primer so that a XmnI restriction site was present in the 1G amplicon (24). PCR was performed in a 50-µl reaction volume containing 50 ng of genomic DNA, 25 pmol of each primer, 0.2 mM dNTPs, 1 × reaction buffer, 1.5 mM MgCl₂ and 1 unit of Taq polymerase (Invitrogen). The PCR was performed in 35 cycles (5 min at 94 °C, 1 min at 94 °C, 1 min at 57 °C, 1 min at 72 °C, followed by a 7-min extension at 72 °C). A 20-µl aliquot of PCR products was digested with XmnI at 37 °C for 3 h. A 20-µl aliquot of the digest was electrophoresed on a 10% vertical nondenaturing polyacrylamide gel at 150 V for 1 h. The gel was then stained with Vistra Green (Amersham Biosciences, Inc.) and scanned with a fluorimager.

Statistical Analysis

The confidence intervals for the activity of MMP-1 promoter-reporter constructs were calculated (25). The differences among cells with different genotypes with respect to MMP-1 RNA and protein expression were analyzed using the Mann-Whitney U test. Student's t test was used to assess significant differences in demographic characteristics among subjects in the case control study. The chi square test was used to determine the significance of the association between MMP-1 promoter alleles and PPROM. The odds ratio and 95% confidence intervals were also determined.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Cultured Amnion Mesenchymal Cells-- Virtually all of the isolated amnion mesenchymal cells stained positive for vimentin after one passage, whereas only few cells stained for cytokeratin (Fig. 1, A-C). Greater than 95% of cells were vimentin-positive and cytokeratin-negative in the analysis of different isolates. These observations indicate that the preparations used in our experiments were highly enriched for cells from the mesenchymal lineage.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Characterization of amnion mesenchymal cells. Panel A, immunocytochemical staining for vimentin. The brown immunoprecipitate identifies vimentin-positive cells. Panel B, immunostaining for cytokeratin 18. Only a few cells stained for cytokeratin 18. Panel C, control preparation processed without primary antibody. All photomicrographs are at 100×.

Relative Activities of MMP-1 1607 2G and 1G Promoter Alleles-- We examined the activities of the two promoter alleles transfected into various cell hosts. The relative promoter activities of the 2G allele to the 1G allele are shown in Table I. Consistent with the report of Rutter et al. (19), we found that in all cell contexts tested, the 2G allele displayed stronger activity than the 1G allele. The difference was greatest in A2058 melanoma cells and was more than 2-fold higher in amnion-derived cells including WISH cells and amnion mesenchymal cells. When transfected amnion mesenchymal cells were treated with 50 ng/ml PMA, we consistently found a modest increase in 2G promoter activity (20-30%) above the Me₂SO control but no response of the 1G promoter. The modest impact of PMA appears to be because the transfection reagents activate signaling pathways mediating MMP-1 gene transcription.

View this table: [in this window] [in a new window]   Table I Relative activities of MMP-1 promoters with 1607 2G or 1G polymorphism Promoter activities determined from relative luciferase activity units were examined in four different cell hosts. Values presented are the mean ± S.E. from three independent experiments conducted with triplicate cultures.

PMA Treatment Increases Levels of a Nuclear Protein Binding with High Affinity to the 2G Allele Sequence-- We analyzed the binding of nuclear proteins prepared from vehicle-treated and PMA-treated amnion mesenchymal cells to oligonucleotide probes containing either the 1G or 2G at 1607 (Fig. 2). Nuclear extracts from the Me₂SO-treated cells produced two complexes, and the binding of nuclear proteins was equivalent for the 1G and 2G probes. In competition assays, binding to the higher mobility DNA-protein complex (arrow) was suppressed by unlabeled homologous oligonucleotide, but in the case of the 2G probe an oligonucleotide containing an AP-1 binding site without the Ets binding site was much less effective in blocking complex formation. A low mobility DNA-protein complex (arrowhead) was also observed which showed only modest suppression by unlabeled homologous oligonucleotide, with the unlabeled AP-1 site oligonucleotide being a less effective competitor. There was a striking difference between the 2G oligonucleotide compared with the 1G probe in the binding of nuclear proteins extracted from PMA-treated cells, suggesting that this agent stimulates the expression or activity of a specific nuclear protein that preferentially recognizes the 2G allele. The binding of proteins in the PMA-treated cell nuclear extracts to the 2G allele was specific in that the high mobility DNA-protein complex was inhibited by unlabeled homologous probe but not by the AP-1 probe. A consensus Ets binding site oligonucleotide, but not an oligonucleotide with a mutated Ets binding site, suppressed formation of the high mobility complex (Fig. 3). The low mobility DNA-protein complex of the 2G probe was also more prominent with nuclear proteins isolated from PMA-treated cells. Formation of this complex was blocked to some degree by homologous unlabeled oligonucleotide and significantly suppressed by the consensus Ets binding site and mutant Ets binding site oligonucleotides, and to a lesser extent by the unlabeled AP-1 probe. This complex is, therefore, unlikely to be Ets-specific, in contrast to the high mobility complex.

View larger version (117K): [in this window] [in a new window]   Fig. 2.   Electrophoretic mobility shift assays of amnion mesenchymal cell nuclear proteins binding to the oligonucleotides containing MMP-1 1G or 2G alleles. Reactions were carried out with labeled 1G and 2G oligonucleotide probes as described under "Experimental Procedures." The specific activities of the labeled 1G and 2G oligonucleotides were 1.1 × 105 and 1.2 × 105 cpm/ng, respectively. DMSO, dimethyl sulfoxide.

View larger version (78K): [in this window] [in a new window]   Fig. 3.   A consensus Ets binding site oligonucleotide competes for complex formation, but not an oligonucleotide with a mutated Ets binding site. Reactions were carried out with a labeled 2G oligonucleotide probe and nuclear extract from PMA-stimulated cells as described under "Experimental Procedures." Arrows indicate the specific high mobility DNA-protein complex.

The nature of the nuclear protein(s) forming the DNA-protein complexes is not known, but presumably it is a member of the Ets transcription factor family based on studies conducted previously by Rutter et al. (19), who first identified the polymorphism and showed that the 2G allele binds recombinant Ets-1. We conducted experiments with antibodies to Ets-1/Ets-2 and Elk-1, members of the Ets family of transcription factors, to determine whether they would ablate or supershift the complexes. Neither of the antibodies affected the complexes (data not shown). These findings suggest that another member of the Ets family is responsible for the complex formation.

The partial inhibition of the complex formation by the AP-1 probe in our studies is not unexpected because Rutter et al. (19) demonstrated that binding of recombinant Ets-1 to the 2G oligonucleotide probe was enhanced in the presence of c-Jun, indicating a possible cooperative interaction between proteins binding to the Ets and AP-1 sites.

Influence of 1607 MMP-1 Promoter Genotype on Induction of MMP-1 mRNA by PMA-- Quantitative real time PCR was performed to assess the induction of MMP-1 mRNA in amnion mesenchymal cells with different 1607 MMP-1 promoter genotypes in response to PMA. The activation ratio for MMP-1 mRNA expression, representing the PMA-induced MMP-1 mRNA abundance divided by the MMP-1 abundance in vehicle-treated cultures, was significantly greater in cells carrying the 2G allele (1G/2G heterozygotes and 2G/2G homozygotes) compared with cells with 1G/1G genotype (cells with 1G/1G genotype, 24.2 ± 9.2 (n = 4); cells with 1G/2G and 2G/2G genotypes, 365.5 ± 153.5 (n = 11); p < 0.01 by the Mann-Whitney U test). Note that the variation in the response of cells resulted in relatively large standard errors in the activation ratios. This probably reflects the contribution of other biological variables affecting MMP-1 expression. Nonetheless, cells with the 1G/2G and 2G/2G genotypes had significantly greater MMP-1 mRNA induction than cells with the 1G/1G genotype. We found no dose relationship between the 2G allele and the induction of MMP-1 mRNA (1G/2G mean activation ratio, 580 ± 2.57; 2G/2G, 100 ± 17.7), suggesting that the 2G allele may exert a dominant effect. Northern blot analysis of RNA extracted from cells with different MMP-1 promoter genotypes confirmed the greater induction of MMP-1 mRNA by PMA in cells with a 2G promoter allele (Fig. 4). Because of the lower sensitivity of Northern analysis, we could not detect MMP-1 mRNA in unstimulated cells.

View larger version (76K): [in this window] [in a new window]   Fig. 4.   PMA stimulation of MMP-1 mRNA and MMP-1 protein accumulation in amnion mesenchymal cells of different genotypes. Panel A, Northern blot analysis of total RNA (10 µg/lane) extracted from amnion mesenchymal cells with the indicated MMP-1 promoter genotype cultured with 2% Me2SO (DMSO) vehicle or 50 ng/ml PMA for 48 h. The membrane was probed with a human MMP-1 cDNA and a human 28 S rRNA probe to assess equality of RNA loading. The ratio of MMP-1 mRNA to 28 S rRNA of PMA-stimulated cells determined by densitometry was 0.22, 2.24, and 3.05 for the 1G/1G, 1G/2G, and 2G/2G genotypes, respectively. Panel B, Western blot analysis of conditioned media from cultures of amnion mesenchymal cells with different genotypes. Equal volumes of media were loaded from cultures with equivalent cell numbers and protein concentrations (2.5 µg/µl) treated with vehicle or PMA as described above.

The differences in PMA-induced MMP-1 mRNA expression in the cells with different MMP-1 promoter genotypes are the result of differences in MMP-1 gene transcription as reflected by the fact that similar differences were observed when nascent transcripts were measured. The activation ratio of MMP-1 nascent transcripts was also greater in mesenchymal cells with a 2G allele (cells with 1G/1G genotype, 23.9 ± 12.6 (n = 4); cells with 1G/2G and 2G/2G genotypes, 215.0 ± 69.5 (n = 6); p < 0.05 by the Mann-Whitney U test).

Influence of 1607 MMP-1 Promoter Genotype on Induction of MMP-1 Protein-- MMP-1 protein production in response to PMA was also influenced by MMP-1 promoter genotype (Fig. 4 and Table II). Cells with a 1G/2G or 2G/2G genotype showed a greater MMP-1 response to PMA stimulation than cells homozygous for the 1G allele in terms of MMP-1 detected by Western blotting and the absolute amount of MMP-1 elaborated as determined by ELISA (p < 0.01). Western blotting revealed that proenzyme (52 kDa) was the predominant form of MMP-1 produced by the PMA-stimulated cells, although the 46-kDa active form of the enzyme was also detected in the conditioned medium of 1G/2G and 2G/2G cells.

View this table: [in this window] [in a new window]   Table II Influence of the 1607 MMP-1 promoter genotype on MMP-1 protein production by amnion mesenchymal cells Quantitative MMP-1 protein analysis was carried out on amnion mesenchymal cells with the indicated MMP-1 promoter genotype. Cells were cultured for 24 h in 2% Me2SO vehicle (Control) or with 50 ng/ml PMA. Values are the means ± S.E. from the indicated number of different cell preparations. MMP-1 production by each cell preparation was determined in triplicate cultures and expressed as secreted MMP-1/µg of cell protein. Values with a different superscript are significantly different (p < 0.01) by the Mann-Whitney U test.

We correlated the induction of MMP-1 mRNA with levels of MMP-1 protein produced in response to PMA treatment in cultures where we had performed both measurements and found a significant correlation (r = 0.847, p = 0.001) between mRNA and protein induction (Fig. 5). The 1G/1G cells had the lowest induction of MMP-1 mRNA and protein.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Correlation between PMA induction of MMP-1 mRNA and protein according to amnion mesenchymal cell genotype. The activation ratio for MMP-1 mRNA representing the PMA-induced MMP-1 mRNA abundance divided by the MMP-1 mRNA levels in vehicle-treated cells is plotted against the level of MMP-1 protein produced by the same cells measured by ELISA as described in Table II. Open circles, cells with a 1G/1G genotype; filled triangles, 1G/2G genotype; open squares, 2G/2G genotype.

Association of the 1607 MMP-1 Polymorphism and PPROM-- There is evidence that preterm birth, and PPROM in particular, is influenced by genetic factors. Evidence consistent with this notion includes the observation that a preterm birth is a strong predictor of subsequent preterm birth, especially when the prior preterm birth was associated with PPROM. African Americans have a higher incidence of preterm birth and PPROM (4). Our recent finding that a polymorphism in the maternal TNFA gene (26) is associated with PPROM also supports this idea. Because of the postulated role of MMPs in fetal membrane rupture, we carried out a case control study examining the association between the fetal genotype at the 1607 MMP-1 promoter polymorphic site and PPROM. The study was conducted on African Americans because of the higher incidence of PPROM in this population.

There were no statistically significant differences in maternal age, gravidity, and parity between the PPROM and the control groups (Table III). As expected, there were statistically significant differences in gestational age at birth and birth weight in these two groups. In the PPROM group, the gestational age at delivery was 7 weeks shorter, and birth weight was 1378 g lower than in the control group.

We found a significant difference in allele and genotype frequencies of the neonates for the 1607 MMP-1 promoter polymorphism between patients with PPROM and those in the control group (Table IV). In the PPROM group the frequency of 1G/2G heterozygotes and 2G/2G homozygotes was significantly higher than in the control group (p = 0.028; odds ratio 2.29, 95% confidence interval, 1.09-4.82). This indicates that a fetus carrying at least one 2G allele is more susceptible to PPROM than a fetus that is homozygous for 1G allele. Conversely, fetuses homozygous for the 1G MMP-1 promoter allele are at a reduced risk for PPROM. The distribution of 1607 MMP-1 promoter genotypes has not been described previously in African American populations. Our distribution of genotypes differs from that reported by Rutter et al. (19) for a Center d'Etude du Polymorphisme Humain (CEPH) population and by Kanamori et al. (27) for a Japanese population. The CEPH sample had a genotype distribution of 31% 1G/1G, 30% 1G/2G, and 39% 2G/2G; the Japanese population had a distribution of 20% 1G/1G, 37.3% 1G/2G, and 42.7% 2G/2G compared with 23.8% 1G/1G, 47.2% 1G/2G, and 28.9% 2G/2G in our study. Although the significance of the different genotype distributions in these three populations to the risk of PPROM cannot be determined at this time, the higher percentage of 1G/1G genotypes in the CEPH data set is consistent with the lower incidence of PPROM in Caucasians.

View this table: [in this window] [in a new window]   Table IV Carrier rates for MMP-1 promoter 1607 alleles among PPROM and control subjects

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our observations document that the insertion of a guanine nucleotide (G) at 1607 in the MMP-1 promoter, shown previously to create an Ets binding site and increased promoter activity in transfected cells (19) and to be associated with increased levels of MMP-1 mRNA in ovarian cancer cells (27), is associated with increased transcription of the endogenous MMP-1, resulting in increased MMP-1 mRNA levels and increased MMP-1 protein production in amnion mesenchymal cells. The evidence for increased MMP-1 transcription is based on quantification of unprocessed MMP-1 RNA, representing nascent MMP-1 transcripts. This is the first direct correlation between the 1607 MMP-1 polymorphism and MMP-1 transcription. Because MMP-1 transcript and protein production correlated with the genotype of the amnion mesenchymal cells and there was no significant difference between the cells with one or two 2G alleles, we concluded that the 2G allele exerts a dominant effect, rendering amnion cells significantly more responsive to PMA-induced MMP-1 expression.

The increased transcription of the endogenous 2G allele is presumably driven by the binding of a transcription factor whose level or activity is increased by PMA. The PMA-induced factor is likely to be a member of the Ets family of transcription factors (28). However, because Rutter et al. (19) found that binding of recombinant Ets-1 to an oligonucleotide containing 2G at 1607 was increased in the presence of c-Jun, it is possible that the augmented binding of nuclear proteins to the 2G allele we observed is the result of PMA induction of c-Jun or another Jun protein.

The impact of the 1607 MMP-1 polymorphism on cellular functions in vivo has been explored in case control studies in women with gynecologic cancers which revealed that ovarian and endometrial cancers (27, 29) are associated with the 2G allele. Rutter et al. (19) also found that a greater percentage of melanoma cell lines were homozygous for the 2G allele compared with CEPH controls (19). The authors of these studies proposed that increased MMP-1 expression by cancer cells would promote tumor metastasis by endowing the cancerous cells with greater capacity to degrade extracellular matrix. We reasoned that the 2G allele would also promote increased breakdown of interstitial collagen of the amnion and thus predispose to PPROM. We assumed that the mesenchymal cells (fibroblasts) of the amnion would be important players in the interstitial collagen breakdown, and therefore we looked for an association between fetal MMP-1 genotype and PPROM. Our findings provide the first evidence for an effect of the 1607 MMP-1 promoter polymorphism on the function of normal (nonmalignant) cells.

Our case control study revealed a significant, albeit modest, association between the fetal 1607 MMP-1 promoter genotype and PPROM. The fact that the association was not more striking undoubtedly reflects the complexity of the genetic and environmental factors that result in PPROM. For example, the activity of MMP-1 is influenced by the level of endogenous tissue inhibitor of metalloproteinase expression (30), the extent of activation of the proenzyme (9, 11, 12), in addition to the level of pro-MMP-1 expression. Thus, multiple events under the control of other genes are required for expression of MMP-1 catalytic activity. Our findings suggest that the functional differences of the two MMP-1 promoter alleles is exposed when cells are challenged with agents that promote MMP expression. Therefore, the 1607 MMP-1 genotype may only have an impact in vivo under specific environmental conditions in which amnion cells are confronted with MMP-1-inducing stimuli (e.g. infection or inflammation). Because other MMPs participate in both the activation of pro-MMP-1 as well as the further catabolism of collagen fragments, variants in other MMP genes may augment or attenuate the influence of the 1607 MMP-1 promoter polymorphism. It will, therefore, be important to explore in the future the relationship between MMP-1 promoter genotype and environment (e.g. chorioamnionitis or bacterial vaginosis) interactions.

Association studies can be confounded by population stratification, a particular concern in studies on heterogeneous populations such as African Americans. Because linkage cannot be established in this type of study design, it is possible that the significant association we found between 1607 MMP-1 promoter polymorphism and PPROM results from the effect of another gene near the MMP-1 gene. The MMP-1 gene on chromosome 11 (31, 32) is located in a cluster of other MMP genes (MMP-3, 7, 8, 10, 12, 13, 20, 26) (33-41). Therefore, it is possible that the association we detected is not the result of a functional role of MMP-1 but of one of the other matrix-degrading enzymes in this gene cluster. However, the results of the in vitro studies we conducted are clearly consistent with the effect being attributed to MMP-1.

We conclude that the 1607 MMP-1 promoter polymorphism has functional significance in MMP-1 transcription in vitro and that this polymorphism makes a significant, although modest, contribution to the risk of PPROM in the African American population.

	ACKNOWLEDGEMENTS

We thank Drs. Joni L. Rutter and Constance E. Brinckerhoff (Dartmouth Medical School) for the MMP-1 promoter fragment used to construct reporter constructs and Judith Wood for assistance in preparation of this manuscript.

	FOOTNOTES

^* This research was supported by National Institutes of Health Grant HD34612 (to J. F. S. III) and by a grant from the Bill and Melinda Gates Foundation (to J. F. S. III).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.

^** To whom correspondence should be addressed: 1354 BRB II, 421 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-0147; Fax: 215-573-5408; E-mail: jfs3@mail.med.upenn.edu.

Published, JBC Papers in Press, December 11, 2001, DOI 10.1074/jbc.M107865200

	ABBREVIATIONS

The abbreviations used are: PPROM, preterm premature rupture of the membranes; MMP, matrix metalloproteinase; AP-1, activator protein-1; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Me₂SO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This article has been cited by other articles:

				H. Wang, M. Ogawa, J. R. Wood, M. S. Bartolomei, M. D. Sammel, J. P. Kusanovic, S. W. Walsh, R. Romero, and J. F. Strauss III Genetic and epigenetic mechanisms combine to control MMP1 expression and its association with preterm premature rupture of membranes Hum. Mol. Genet., April 15, 2008; 17(8): 1087 - 1096. [Abstract] [Full Text] [PDF]

				J. V. Cockle, N. Gopichandran, J. J. Walker, M. I. Levene, and N. M. Orsi Matrix Metalloproteinases and Their Tissue Inhibitors in Preterm Perinatal Complications Reproductive Sciences, October 1, 2007; 14(7): 629 - 645. [Abstract] [PDF]

				H. Wang, S. Parry, G. Macones, M. D. Sammel, H. Kuivaniemi, G. Tromp, G. Argyropoulos, I. Halder, M. D. Shriver, R. Romero, et al. From the Cover: A functional SNP in the promoter of the SERPINH1 gene increases risk of preterm premature rupture of membranes in African Americans PNAS, September 5, 2006; 103(36): 13463 - 13467. [Abstract] [Full Text] [PDF]

				K. H. Taylor, R. D. Schnabel, and J. F. Taylor Impaired collagen chaperone results in preterm PROM PNAS, September 5, 2006; 103(36): 13267 - 13268. [Full Text] [PDF]

				K. Shan, W. Ying, Z. Jian-Hui, G. Wei, W. Na, and L. Yan The function of the SNP in the MMP1 and MMP3 promoter in susceptibility to endometriosis in China Mol. Hum. Reprod., June 1, 2005; 11(6): 423 - 427. [Abstract] [Full Text] [PDF]

				H. Wang, S. Parry, G. Macones, M. D. Sammel, P. E. Ferrand, H. Kuivaniemi, G. Tromp, I. Halder, M. D. Shriver, R. Romero, et al. Functionally significant SNP MMP8 promoter haplotypes and preterm premature rupture of membranes (PPROM) Hum. Mol. Genet., November 1, 2004; 13(21): 2659 - 2669. [Abstract] [Full Text] [PDF]

				K. Hao, X. Wang, T. Niu, X. Xu, A. Li, W. Chang, L. Wang, G. Li, N. Laird, and X. Xu A candidate gene association study on preterm delivery: application of high-throughput genotyping technology and advanced statistical methods Hum. Mol. Genet., April 1, 2004; 13(7): 683 - 691. [Abstract] [Full Text] [PDF]

				C. A. Wyatt, C. I. Coon, J. J. Gibson, and C. E. Brinckerhoff Potential for the 2G Single Nucleotide Polymorphism in the Promoter of Matrix Metalloproteinase to Enhance Gene Expression in Normal Stromal Cells Cancer Res., December 15, 2002; 62(24): 7200 - 7202. [Abstract] [Full Text] [PDF]

				P. E. Ferrand, T. Fujimoto, V. Chennathukuzhi, S. Parry, G. A. Macones, M. Sammel, H. Kuivaniemi, R. Romero, and J. F. Strauss III The CARD15 2936insC mutation and TLR4 896 A>G polymorphism in African Americans and risk of preterm premature rupture of membranes (PPROM) Mol. Hum. Reprod., November 1, 2002; 8(11): 1031 - 1034. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/8/6296    most recent M107865200v1