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Matrix metalloproteinase-8 is expressed in human chorion during labor
American Journal of Obstetrics and Gynecology (2004) 190, 843-50
www.elsevier.com/locate/ajog
Matrix metalloproteinase-8 is expressed in human chorion during labor Fabia´n Arechavaleta-Velasco,PhD,a Dominic Marciano, MD,b Laura Dı´az-Cueto, MD, PhD,a Samuel Parry, MDb Research Unit in Reproductive Medicine, Hospital de Ginecobstetricia ‘‘Luis Castelazo Ayala’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico,a and the Center for Research on Reproduction and Women’s Health, University of Pennsylvania, Philadelphia, Pab Received for publication December 20, 2002; revised May 21, 2003; accepted September 19, 2003
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Parturition Neutrophil collagenase Matrix metalloproteinase-8 Fetal membrane
Objective: The purpose of this study was to investigate the expression of matrix metalloproteinase-8 by human fetal membranes during labor. Study design: Fetal membranes were obtained from women who underwent normal labor or elective cesarean delivery at term. Matrix metalloproteinase-8 levels in fetal membranes were determined by enzyme-linked immunosorbent assay and Western blot; the expression of the matrix metalloproteinase-8 gene was detected by reverse transcriptionepolymerase chain reaction. Immunohistochemistry and in situ hybridization was performed to localize matrix metalloproteinase-8 protein and messenger RNA in intact membranes. Results: Matrix metalloproteinase-8 protein levels were increased 5-fold in fetal membranes from labor compared with membranes that were obtained from cesarean delivery. Western blots confirmed the presence of matrix metalloproteinase-8 in protein extracts. Reverse transcriptionepolymerase chain reaction, in situ hybridization, and immunohistochemistry demonstrated that matrix metalloproteinase-8 messenger RNA and protein were expressed almost exclusively in the chorion after labor. Conclusion: We conclude that matrix metalloproteinase-8 is produced primarily by chorionic cells in human fetal membranes and that the level of matrix metalloproteinase-8 protein and messenger RNA expression in fetal membranes increases during labor. Ó 2004 Elsevier Inc. All rights reserved.
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Supported by a grant from the Bill and Melinda Gates Foundation and by a grant (D43-TW-00671) from the Fogarty International Center (F.A-V.). Reprint requests: Center for Research on Reproduction and Women’s Health, University of Pennsylvania, School of Medicine, 1352 Biomedical Research Building II/III, 421 Curie Blvd, Philadelphia, PA 19104-6142. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.09.032
The primary role of the amniochorion, or fetal membranes, is to provide the fetus with a selective barrier against pathogenic organisms throughout gestation. Secondary functions include the synthesis of amniotic fluid, the maintenance of immune tolerance, and the secretion of hormones, neuropeptides, and cytokines.1 Premature rupture of the membranes (ie, rupture of the amniochorion preceding labor) often results in perinatal and neonatal infections; preterm premature rupture
844 of the membranes (ie, !37 weeks of gestation) is the leading identifiable cause of spontaneous preterm delivery.2 Unfortunately, the precise nature of the structural alterations that predispose the fetal membranes to rupture and the signals that regulate the onset of such changes remains poorly characterized. The activation of collagenolytic enzymes has been postulated to be a labor-associated event that leads to the degradation of amniochorion connective tissue and weakening of the membranes. An active connective tissue-degrading system has been demonstrated in fetal membranes under physiologic conditions, and the activity of these enzymes has been correlated with labor.3-5 Studies of proteolytic activities in fetal membranes and amniotic fluid revealed that, under normal conditions, a complex network of matrix metalloproteinases (MMPs) and their inhibitors6,7 controls the catabolism of extracellular matrix components. Several MMPs, which include MMP-2, MMP-3, and MMP-9, have been identified in human amniochorion.6,8 However, the predominant collagen in fetal membranes that is responsible for their tensile strength is collagen type I, which is degraded by the collagenases MMP-1 and MMP-8.9 The expression of MMP-1, which is produced by numerous cell types, is increased in the fetal membranes before labor,10 and levels of MMP-8 are increased in amniotic fluid of women with spontaneous term labor7,11 and in cases of intra-amniotic infection.7,12 Although MMP-8 was thought previously to be expressed exclusively by neutrophils, recent evidence has suggested that different cell types produce this MMP, including chondrocytes, fibroblasts, bronchial epithelial cells, and endothelial cells.13-15 Because the significance of MMP-8 in fetal membrane rupture is unknown, we sought to investigate the expression of MMP-8 by human fetal membranes before and after labor. The results from this study indicate that both MMP-8 protein and messenger RNA (mRNA) are produced by chorionic cells and that the increased production of MMP-8 by these cells may be important in fetal membrane rupture and labor.
Material and methods Biologic samples Fetal membranes were obtained from 2 groups of women: pregnant women (n = 7) with uncomplicated spontaneous labor at term and women (n = 7) with obstetric indications for elective cesarean delivery at term, without labor, and no associated pregnancy complications. No significant differences were observed in the gestational ages between both groups. Infection was excluded by clinical parameters. Tissue that was obtained from the midzone of the fetal membranes was processed immediately after deliv-
Arechavaleta-Velasco et al ery. The fetal membranes were dissected free of the placenta and were washed of all blood with the use of sterile heparinized saline solution. Adherent decidual tissue was removed with sterile cotton gauze according to the method described by Fortunato et al.16 For immunohistochemistry and in situ hybridization, samples were fixed in 10% buffered formalin overnight, washed in 50 mmol/L Tris buffer pH 7.5 that contained 0.15 mol/ L NaCl, and embedded in paraffin wax.
Preparation of amnion and chorion extracts After the maternal surface of the chorion was scraped and washed with heparinized saline solution to remove any decidual cells or blood, the fetal membranes were identified and separated manually into amnion and chorion. One to 2 g of amniochorion, amnion, or chorion were homogenized with a Polytron (Brinkmann, Westbury, NY) in 2 volumes of 50 mmol/L Tris buffer pH 7.4 that contained 0.15 mol/L NaCl, Triton X-100, and 100 mmol/L CaCl2, according to the method of Woessner and Taplin.17 Protein concentration was measured according to Bradford’s method.18
MMP-8 assay MMP-8 protein levels in amniochorion, amnion, and chorion extracts were determined with the BioTrak MMP-8 (human MMP-8, neutrophil collagenase) enzyme-linked immunosorbent assay (ELISA) system (Amersham Pharmacia Biotech, Piscataway, NJ), which is based on a 1-step sandwich enzyme-linked immunosorbent assay, according to the manufacturer’s protocol. Briefly, a 100 mL volume of the standard or the samples was transferred to a 96-well microtiter plate that had been coated previously with mouse anti-human MMP8 monoclonal antibody. The plate was incubated for 2 hours at room temperature. After being washed 3 times, the wells were incubated for 1 hour at room temperature with 100 mL of an anti-human MMP-8 polyclonal antibody that was conjugated with peroxidase. The wells were washed again 3 times; 100 mL of tetramethylbenzidine substrate solution was added, and the reaction was allowed to proceed for 30 minutes. The resulting color intensity was measured at 450 nm in an ELISA reader. Each sample was analyzed 3 times.
Western blots Western blotting was performed to confirm ELISA results and to determine whether precursor or active MMP-8 was present in the fetal membranes. Twenty micrograms of protein from amniochorion, amnion, or chorion extracts were separated by 8% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). Primary monoclonal antibody against MMP-8 (R&D Systems, Minneapolis,
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Arechavaleta-Velasco et al Table I
MMP-8 and GAPDH primer and probe sequences
Primer name
Sequence
RT-PCR MMP-8 forward MMP-8 reverse GAPDH forward GAPDH reverse
5#-ATG GAC CAA CAC CTC CGC AA-3# 5#-GTC AAT TGC TTG GAC GCT GC-3# 5#-CTG AGA ACG GGA AGC TTG TCA TCA A-3# 5#-GCC TGC TTC ACC ACC TTC TTG ATG TC-3#
Real-time RT-PCR MMP-8 forward MMP-8 reverse GAPDH forward GAPDH reverse
5#-GTG ACC CCA GTT TGA CAT TTG AT-3# 5#-TCT TTG TAG CTG AGG ATG CCT TC-3# 5#-CCC CTT CAT TGA CCT CAA CTA CA-3# 5#-CGC TCC TGG AAG ATG GTG AT-3#
In situ hybridization Sense Antisense
5#-GGC ATT CAG GCC ATC TAT GGA CTT TCA AGC AAC CCT ATC C-3# 5#-GGA TAG GGT TGC TTG AAA GTCCAT AGA TGG CCT GAA TGC C-3#
Minn) was used at 2 mg/mL and incubated with the blot overnight at 4(C. The primary antibody was detected with the use of SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill). MMP-8 that was purified from stimulated neutrophils (Calbiochem, San Diego, Calif) was used as a positive control.
Immunohistochemical determination of MMP-8 Paraffin-embedded tissues were deparaffinized and rehydrated with xylene and graded alcohol. Sections were blocked with 10% normal horse serum for 1 hour at room temperature. Subsequently, mouse monoclonal antibody raised against MMP-8 (R&D Systems) was applied at 25 mg/mL for 1.5 hour at 37(C, followed by detection with the Vectastain ABC-alkaline phosphatase anti-mouse kit (Vector Laboratories, Burlingame, Calif) and the Vector Red substrate kit (Vector Laboratories). Controls for immunohistochemistry experiments included the use of mouse immunoglobulin G as the primary antibody and the omission of primary antibody (secondary antibody alone).
Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from 500 mg of fresh tissue by TRIzol reagent (Invitrogen, Carlsbad, Calif). After RNase-free DNase (Invitrogen) treatment for 15 minutes at room temperature, RNA was further purified with RNeasy RNA isolation kit (QIAGEN, Valencia, Calif). Total RNA (2 mg) was reverse transcribed in a 20 mL reaction system with the superscript 1-step RT-PCR system (Invitrogen), according to the manufacturer’s protocol. Reverse-transcribed complementary DNA (2 mL) was amplified in a 25 mL PCR system that contained 200 mmol/L each deoxyribonucleoside triphosphate, 300 nmol/L of each primer, standard buffer supplemented
with 1 unit of platinum Taq DNA polymerase (Invitrogen), and 1.5 mmol/L MgCl2. After an initial denaturation step at 94(C for 4 minutes, 30 cycles of PCR were performed with denaturation at 94(C for 30 seconds, annealing at 56(C for 30 seconds, and extension at 72(C for 45 seconds. The last extension was performed at 72(C for 7 minutes. Specific primers (not recognizing MMP-1 or other MMPs) were designed on the basis of the published DNA sequence to amplify a 531ebase pair segment of the MMP-8 gene between nucleotides 674 and 1205 (Table I).19 Primers were also constructed for the constitutively expressed housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).19,20 Sterile water was included as a negative control in each PCR experiment.
Quantification of MMP-8 transcripts by real-time RT-PCR The mRNA level of MMP-8 was quantified by real-time RT-PCR in the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif). PCR was performed with SYBR Green PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. Quantitative PCR primers are depicted in Table I. PCR cycles consisted of an initial denaturation step at 95(C for 10 minutes, followed by 40 cycles at 95(C for 15 seconds and 60(C for 60 seconds. PCR amplification of the housekeeping gene GAPDH was done for each sample as a control for sample loading and to allow normalization between samples. A standard curve was constructed with pCR-XL-TOPO vector (Invitrogen) that contained the same fragment that was amplified by the SYBR Green PCR master mix. The relative expression in each sample was calculated with respect to the standard calibration curve, as previously described.21,22 Each sample was analyzed twice; each
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Arechavaleta-Velasco et al 250 mg/mL yeast transfer RNA, 5 mg/mL polydeoxyadenylic acid,100 mg/mL polyadenylic acid, 0.05 pM/mL randomer oligonucleotide, and 500 mg/mL denatured and sheared salmon sperm DNA). Several washes at 37(C were carried out with SSC in constant agitation (2 ! SSC for 15 minutes, 1 ! SSC for 15 minutes, 0.5 ! SSC for 15 minutes). Digoxigenin was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments and thereafter by staining with NBT/ BCIP chromogen (Roche Molecular Biochemicals).
Statistical analysis Results are presented as mean G SEM of MMP-8 concentration or number of copies (MMP-8/1000 copies of GAPDH). Comparisons between groups were performed by Mann-Whitney rank sum test. A probability value of !.05 was considered to be the limit for statistical significance.
Results
Figure 1 Concentration of MMP-8 protein in (a) amniochorion, and (b) amnion and chorion extracts that were obtained from normal labor and cesarean delivery (C/S). MMP-8 levels were quantified by ELISA. Each bar represents the meanGSEM of 7 samples, which were measured in duplicate. Statistical analysis by Mann-Whitney rank sum test revealed that MMP-8 levels were significantly different (P!.05). The asterisk indicates a statistically significant difference.
PCR experiment included 2 nonetemplate control wells. PCR products were confirmed as single bands with gel electrophoresis.
In situ hybridization Specific sense and antisense probes for MMP-8 (not recognizing other MMPs) were designed, on the basis of the published complementary DNA sequence (Table I).19 These probes were labeled with digoxigenin-conjugated deoxyuridine triphosphate with the DIG oligonucleotide 3#-end labeling kit (Roche Molecular Biochemicals, Indianapolis, Ind). Six-micron sections of paraffin-embedded amniochorion that were obtained from normal labor were processed for in situ hybridization with the digoxigenin-labeled probes. Hybridization was performed at 57(C overnight in a humidified chamber with 30 ng of digoxigenin-labeled oligonucleotide probe in the hybridization solution (2 ! sodium saline citrate [SSC], 1 ! Denhardt’s solution, 10% dextran sulfate, 50 mmol/ L phosphate buffer pH 7.0, 50 mmol/L dithiothreitol,
The levels of MMP-8 were determined by ELISA in the amniochorion extracts that were obtained from normal labor and cesarean delivery. A wide standard deviation was observed in the total amount of protein that was extracted from the amniochorion. Therefore, ELISA results were normalized against the protein concentration. Enzyme immunoassay of MMP-8 revealed concentration differences between the 2 groups (Figure 1, a). Amniochorion extracts that were obtained from normal labor were increased significantly (n = 7 pregnancies; 198.80 G 26.37 ng/mg total protein) compared with those extracts that were obtained from elective cesarean delivery (n = 7 pregnancies; 40.84 G 6.77 ng/mg total protein; P ! .001). To determine which layer within the fetal membranes contained MMP-8 protein, extracts from the chorion and amnion were analyzed separately by ELISA. MMP-8 was detectable in chorion and amnion from elective cesarean delivery and labor. However, significantly higher concentrations of MMP-8 were detected in the chorion than the amnion (Figure 1, b). The levels of MMP-8 were increased significantly in chorion that was obtained after normal labor (n = 7 pregnancies; 116.0 G 12.33 ng/mg total protein), compared with chorion samples that were obtained after elective cesarean delivery (n = 7 pregnancies; 51.78 G 10.14 ng/mg total protein; P ! .001). There were no significant differences in MMP-8 concentrations between the amnion from normal labor (n = 7 pregnancies; 5.66 G 1.37 ng/mg total protein) and from elective cesarean delivery (n = 7 pregnancies; 3.81 G 1.13 ng/mg total protein). Western blots containing protein that was extracted from the same fetal membrane samples were performed to confirm the ELISA results. A representative
Arechavaleta-Velasco et al
Figure 2 Immunoreactive MMP-8 in human amniochorion. Extracts (a) from amnion (Am), chorion (Ch), and amniochorion (ACh) that were collected from cesarean delivery (C/S) or normal labor and (b) from PMN or amniochorion extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting with a murine monoclonal antibody against MMP-8. a, Amniochorion, amnion, and chorion proeMMP-8 at 65 kd and active MMP-8 at 50 kd. b, Purified human neutrophil MMP-8 with proeMMP-8 at 75 kd and active enzyme at 65 kd and amniochorion proenzyme and active enzyme at 65 and 50 kDa, respectively.
Western blot is shown in Figure 2, a. Bands at 65 and 50 kd, which correlated to proMMP-8 and MMP-8, respectively, were found in the amniochorion and chorion extracts, but not in the amnion extract that was obtained from normal labor and cesarean delivery. Molecular weight differences were observed in MMP-8 bands with protein extracts from polymorphonuclear neutrophils (PMNs) and amniochorion (Figure 2, b). The PMN extracts yielded bands at 85, 75, and 65 kd, which corresponded to preproMMP-8, proMMP-8, and active MMP-8, respectively.23,24 The 50-kd band in amniochorion and chorion most likely represents the nonglycosylated form of MMP-8.14,25 Even though immunoreactive proMMP-8 and MMP-8 were found in extracts of amniochorion and chorion collected before labor, the levels of the enzymes were always substantially lower than those detected after delivery. The findings of several Western blots confirmed that proMMP-8 and MMP-8 concentrations were increased in amniochorion and chorion extracts that were obtained from normal labor, compared with those samples obtained from cesarean delivery. MMP-8 mRNA expression in amniochorion, chorion, and amnion that was obtained from normal labor
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Figure 3 MMP-8 mRNA expression in human fetal membranes. Total RNA isolated from human amniochorion that was obtained from (a) cesarean delivery (C/S) and normal labor and from (b) delivered amnion (Am) or chorion (Ch) were transcribed into cDNA by reverse transcription. MMP-8 transcripts were amplified by PCR with specific MMP-8 oligonucleotides and subjected to agarose gel electrophoresis. PCR amplification of the housekeeping gene GAPDH was done for each sample as a control for sample loading. Realtime RT-PCR shows an incremental increase in MMP-8 expression in the amniochorion (c), amnion, and chorion (d) that were obtained from normal labor compared with those obtained from cesarean delivery (C/S). Each bar represents the mean G SEM of number of copies of MMP-8 per 1000 copies of GAPDH of 7 samples. With statistical analysis by MannWhitney rank sum test, MMP-8 mRNA levels were significantly different (P!.05). The asterisk indicates statistical significant difference.
and cesarean delivery was analyzed initially by RTPCR. This analysis revealed that MMP-8 mRNA was expressed in amniochorion that was obtained from normal labor and in the tissues that were collected from cesarean delivery (Figure 3, a). When amnion and chorion were separated manually, MMP-8 mRNA was expressed primarily by the chorion (Figure 3, b). To quantify the expression levels of MMP-8 mRNA, real-time RTPCR was performed with the same tissues. Amniochorion and chorion membranes that were obtained from normal labor showed a significant increase (P = .03) of MMP-8 mRNA expression, compared with those samples that were obtained from cesarean delivery (Figure 3, c and d). In contrast with the qualitative RT-PCR, the increased sensitivity of this technique permitted us to show that the amnion that was obtained from normal labor expressed low levels of the MMP-8 mRNA (Figure 3, d). No band was observed with the negative control (data not shown).
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Figure 4 Immunohistochemical localization of MMP-8 in fetal membranes. Section of fetal membranes were obtained from cesarean delivery (a: original magnification, !10; c: original magnification, !20) and normal labor (b: original magnification, !10; d: original magnification, !20). MMP-8 is located mainly in chorionic cells in fetal membranes that were obtained from normal labor (b, d). The arrows point to cells with positive staining. The arrowheads point to the amnion.
To localize MMP-8 protein in amniochorion membranes, immunohistochemistry with antieMMP-8 antibody was carried out. The expression of MMP-8 protein was observed in the chorionic layer of the membranes that were obtained from normal labor and cesarean deliveries (Figure 4). Immunoreactive protein was distributed homogeneously within the cell cytoplasm. The fetal membranes from patients with normal labor demonstrated a higher intensity of staining for this enzyme. Faint staining was observed in the amnion cells of the samples that were obtained from normal labor (Figure 4, a). No positive cells were detected in the amnion of fetal membranes that were obtained from cesarean delivery (Figure 4, b). Immunoreactive protein in the amnion was present mainly on the surface of the amnion cells. These results were consistent in the 7 samples from each group that were analyzed. Neither control (goat immunoglobulin G as the primary antibody or secondary antibody alone) resulted in any appreciable staining of the amniochorion (data not shown). To localize the exact site and cellular distribution of MMP-8 mRNA expression in amniochorion that was obtained from women after labor at term, in situ hybridization was performed. Similar to the immunohisto-
Arechavaleta-Velasco et al
Figure 5 Localization of MMP-8 transcripts in human amniochorion that was collected from normal labor by in situ hybridization histochemistry. Fetal membranes were obtained from normal labor and hybridized with the MMP-8 antisense probe (a: original magnification, !10; c: original magnification, !20). The red reaction product indicates the presence of MMP-8 mRNA in the chorionic cells. No signals were detectable in parallel sections that were hybridized with the MMP-8 sense probe (b: original magnification, !10; d: original magnification, !20).
chemistry findings, MMP-8 in situ hybridization signals with the antisense probe were located mainly in the chorionic cells of membranes that were collected after labor (Figure 5, a and c). Hybridization signals were not detected when a sense probe was used (negative control; Figure 5, b and d).
Comment MMPs have been implicated in the mechanisms that lead to membrane rupture during labor.2,6,8,26 Human collagenases (MMP-1, MMP-8, and MMP-13) share the unique ability to cleave the triple helical domain of fibrillar collagen types I, II, and III, which form the backbone of the extracellular matrix of the fetal membranes and are responsible for most of the amnion tensile strength.9,27 MMP-8 is regarded as a neutrophilspecific MMP that is stored in granules and released on cell activation.27 This study shows, for the first time, that MMP-8 is expressed by chorionic cells in the human fetal membranes. The expression of MMP-8 was demonstrated with the use of Western blotting and RT-PCR. The predominant form of active MMP-8 in amniochorion
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Arechavaleta-Velasco et al and chorion migrated on sodium dodecylsulfate-polyacrylamide gel electrophoresis at a size of 50 kd and appears to be generated from a 65-kd latent MMP-8, which is in contrast with the 65-kd active and 85-kd latent forms of MMP-8 that were detected in amniotic fluid specimens that were obtained by amniocentesis from women with intra-amniotic infection.12 Although there is a discrepancy in the sizes of active and latent MMP-8 that were obtained from amniotic fluid and fetal membrane extracts, the MMP-8 species that were observed by immunoblotting in our study are comparable to the molecular masses of latent and active MMP-8 that were reported previously in other cell types.14,25 In addition, the direct comparison of PMN and amniochorion MMP-8 shows that the molecular weights of both types of the enzyme are different. These differences may be due to the degree of protein glycosylation between the chorionic and neutrophilic MMP-8. Enzyme-linked immunosorbent assay, but not Western blot, revealed that MMP-8 was present in the amnion; this discrepancy may be due to the sensitivity between the 2 techniques. Immunocytochemical localization revealed that the MMP-8 was located mainly on the surface of amnionic cells. Real-time RT-PCR results revealed that MMP-8 mRNA was expressed at low levels in this tissue. These results raise the possibility that most of the MMP-8 that is present in the amnion might be translocated to the surface of the amnionic cells after being synthesized in the chorionic cells. A similar observation was made previously with the secretion of MMP-2 and MMP-9 in a 3-dimensional co-culture of normal endometrial stromal cells and endometrial cancer cells.28 The striking increase in MMP-8 that we observed during labor appears to be a consequence of both increased MMP-8 mRNA expression and MMP-8 protein synthesis. The sites of MMP-8 synthesis in the amniochorion were determined by immunohistochemistry and in situ hybridization. In the fetal membranes that were obtained after labor, MMP-8 protein and mRNA were co-localized in the chorionic cells. Our findings are congruent with previous reports that demonstrated that MMP-8 concentrations in amniotic fluid are elevated during spontaneous term labor.7 It is possible that neutrophils are recruited and activated in the membranes, uterine wall, and cervical stroma during parturition.29 However, our data show that the chorionic cells, in combination with neutrophils, may be responsible for the MMP-8 increment. The increase in MMP-8 in the amniochorion provides a mechanism for the catabolism of collagen type I, the main collagen of fetal membranes.9 The denatured collagen type I fragments and collagen type IV are substrates for MMP-9, which are also increased in the amniochorion during labor.8 Thus, the combined actions of MMP-8, MMP-9, and other MMPs may provide a mechanism for weakening of the fetal membranes during labor.
The collagenolytic process in fetal membranes that is associated with normal labor is due to an increase of MMPs under the influence of physiologic signals, such as platelet-activating factor, endothelins, prostaglandins, and cytokines.30-32 Although we did not study MMP-8 regulation, previous reports establish that MMP-8 can be induced in other tissues by inflammatory cytokines.13,14 Therefore, we hypothesize that, as with other members of the MMP family in the amniochorion,32 the expression of MMP-8 could be regulated by tumor necrosis factorea and interleukin-1b. Additional studies that will investigate the factors that induce MMP-8 expression in fetal membranes and the expression of MMP-8 in these tissues in cases of intra-amniotic infection are warranted. In summary, MMP-8 was considered previously to be expressed exclusively by neutrophils. The results of the present study show that chorionic cells are able to express MMP-8 mRNA and protein in vivo. The fact that chorionic cells express MMP-8 suggests that MMP-8 is one of the proteinases in amniochorion that are responsible for degrading collagen type I, which results in a progressive loss of amniochorion tissue strength that may lead to its breakdown.
Acknowledgments We thank Dr Jose R. Conejo-Garcia for his help with the real-time RT-PCR technique.
References 1. Petraglia F, Florio P, Napp C, Genazzani AR. Peptide signaling in human placenta and membranes: autocrine, paracrine and endocrine mechanisms. Endocr Rev 1996;17:156-86. 2. Parry S. Strauss JF III. Premature rupture of the fetal membranes. N Engl J Med 1998;338:663-70. 3. Halaburt JT, Uldbjerg N, Helming R, Ohlsson K. The concentration of collagen and the collagenolytic activity in the amnion and the chorion. Eur J Obstet Gynecol Reprod Biol 1989;31:75-82. 4. Bryant-Greenwood GD, Yamamoto SY. Control of peripartal collagenolysis in the human chorion-decidua. Am J Obstet Gynecol 1995;172:63-70. 5. Fortunato SJ, Menon R. Screening of novel matrix metalloproteinases (MMPs) in human fetal membranes. J Assist Reprod Genet 2002;19:483-6. 6. Fortunato SJ, Menon R, Lombardi SJ. Collagenolytic enzymes (gelatinases) and their inhibitors in human amniochorionic membrane. Am J Obstet Gynecol 1997;177:731-41. 7. Maymon E, Romero R, Pacora P, Gomez R, Athayde N, Edwin S, et al. Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, premature rupture of the membranes, and intrauterine infection. Am J Obstet Gynecol 2000;183: 94-9. 8. Vadillo-Ortega F, Gonzalez-Avila G, Furth EE, Hanqin L, Muschel R, Stetler-Stevenson W, et al. 92 kDa type IV collagenase (matrix metalloprotinase-9) activity in human amniochorion increases with labor. Am J Pathol 1995;146:148-56. 9. Bryant-Greenwood G. The extracellular matrix of the human fetal membranes: structure and function. Placenta 1998;19:1-11.
850 10. Vettraino IM, Roby J, Tolley T, Parks WC. Collagenase-I, stromelysin-I, and matrilysin are expressed within the placenta during multiple stages of human pregnancy. Placenta 1996;17:557-63. 11. Maymon E, Romero R, Chaiworapongsa T, Berman S, Conoscenti G, Gomez R, et al. Amniotic fluid matrix metalloproteinase-8 in preterm labor with intact membranes. Am J Obstet Gynecol 2001;185:1149-55. 12. Angus S, Segel S, Hsu CD, Laksmith G, Clark P, Sammel M, et al. Amniotic fluid matrix metalloproteinase-8 indicates intra-amniotic infection. Am J Obstet Gynecol 2001;185:1232-8. 13. Cole AA, Chubinskaya S, Schumacher B, Huch K, Szabo G, Yao J, et al. Chondrocyte matrix metalloproteinase-8: human articular chondrocytes express neutrophil collagenase. J Biol Chem 1996; 271:11023-6. 14. Hanemaaijer R, Sorsa T, Konttinen YT, Ding Y, Swutinen M, Visser H, et al. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells: regulation by tumor necrosis factor-alpha and doxycycline. J Biol Chem 1997; 272:31504-9. 15. Prikk I, Maisi P, Pirila¨ E, Sepper R, Salo T, Wahlgren J, et al. In vivo collagenase-2 (MMP-8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis. J Pathol 2001;194:232-8. 16. Fortunato SJ, Menon R, Swan KF, Lyden TW. Organ culture of amniochorionic membrane in vitro. Am J Reprod Immunol 1994; 32:184-7. 17. Woessner JF, Taplin C. Purification and properties of a small latent matrix metalloproteinase of the rat uterus. J Biol Chem 1988;263:16918-25. 18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 1976;72:248-54. 19. Hasty KA, Pourmotabbed TF, Goldberg GI, Thompson JP, Spinella DG, Stevens RM, et al. Human neutrophil collagenase: a distinct gene product with homology to other matrix metalloproteinases. J Biol Chem 1990;265:11421-4. 20. Ercolani L, Florence B, Denaro M, Alexander M. Isolation and complete sequence of a functional human glyceraldehyde3-phosphate dehydrogenase gene. J Biol Chem 1988;263: 15335-15341. 21. Garcia JR, Krause A, Schulz S, Rodriguez-Jimenez FJ, Kluver E, Adermann K, et al. Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB J 2001;15:1819-21.
Arechavaleta-Velasco et al 22. Garcia JR, Jaumann F, Schulz S, Krause A, Rodriguez-Jimenez J, Forssmann U, et al. Identification of a novel, multifunctional betadefensin (human beta-defensin 3) with specific antimicrobial activity: Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res 2001;306:257-64. 23. Knauper V, Kramer S, Reinke H, Tschesche H. Characterization and activation of procollagenase from human polymorphonuclear leucocytes. N-terminal sequence determination of the proenzyme and various proteolytically activated forms. Eur J Biochem 1990; 189:295-300. 24. Knauper V, Osthues A, DeClerck YA, Langley KE, Blaser J, Tschesche H. Fragmentation of human polymorphonuclearleukocyte collagenase. Biochem J 1993;291:847-54. 25. Hasty KA, Jeffrey JJ, Hibbs MS, Welgus HG. The collagen substrate specificity of human neutrophil collagenase. J Biol Chem 1987;262:10048-52. 26. Athayde N, Edwin SS, Romero R, Gomez R, Maymon E, Pacora P, et al. A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes. Am J Obstet Gynecol 1998;179: 1248-53. 27. Jeffrey JJ. Interstitial collagenases. In: Parks WC, Mecham RP, editors. Matrix metalloproteinases. San Diego (CA): Academic Press; 1998. p. 15-42. 28. Park DW, Ryu HS, Choi DS, Park YH, Chang KH, Min CK. Localization of matrix metalloproteinases on endometrial cancer cell invasion in vitro. Gynecol Oncol 2001;82:442-9. 29. Winkler M, Fischer DC, Ruck P, Marx T, Kaiserling E, Oberpichler A, et al. Parturition at term: parallel increases in interleukin-8 and proteinase concentrations and neutrophil count in the lower uterine segment. Hum Reprod 1999;14:1096-100. 30. Qin X, Garibay-Tupas J, Chua PK, Cachola L, Bryant-Greenwood GD. An autocrine/paracrine role of human decidual relaxin. I: interstitial collagenase (matrix metalloproteinase-1) and tissue plasminogen activator. Biol Reprod 1997;56:800-11. 31. McLaren J, Taylor DJ, Bell SC. Prostaglandin E2-dependent production of latent matrix metalloproteinase-9 in cultures of human fetal membranes. Mol Hum Reprod 2000;6: 1033-1040. 32. Arechavaleta-Velasco F, Ogando D, Parry S, Vadillo-Ortega F. Production of matrix metalloproteinase-9 in lipopolysaccharidestimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system. Biol Reprod 2002;67:1952-8.
www.elsevier.com/locate/ajog
Matrix metalloproteinase-8 is expressed in human chorion during labor Fabia´n Arechavaleta-Velasco,PhD,a Dominic Marciano, MD,b Laura Dı´az-Cueto, MD, PhD,a Samuel Parry, MDb Research Unit in Reproductive Medicine, Hospital de Ginecobstetricia ‘‘Luis Castelazo Ayala’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico,a and the Center for Research on Reproduction and Women’s Health, University of Pennsylvania, Philadelphia, Pab Received for publication December 20, 2002; revised May 21, 2003; accepted September 19, 2003
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Parturition Neutrophil collagenase Matrix metalloproteinase-8 Fetal membrane
Objective: The purpose of this study was to investigate the expression of matrix metalloproteinase-8 by human fetal membranes during labor. Study design: Fetal membranes were obtained from women who underwent normal labor or elective cesarean delivery at term. Matrix metalloproteinase-8 levels in fetal membranes were determined by enzyme-linked immunosorbent assay and Western blot; the expression of the matrix metalloproteinase-8 gene was detected by reverse transcriptionepolymerase chain reaction. Immunohistochemistry and in situ hybridization was performed to localize matrix metalloproteinase-8 protein and messenger RNA in intact membranes. Results: Matrix metalloproteinase-8 protein levels were increased 5-fold in fetal membranes from labor compared with membranes that were obtained from cesarean delivery. Western blots confirmed the presence of matrix metalloproteinase-8 in protein extracts. Reverse transcriptionepolymerase chain reaction, in situ hybridization, and immunohistochemistry demonstrated that matrix metalloproteinase-8 messenger RNA and protein were expressed almost exclusively in the chorion after labor. Conclusion: We conclude that matrix metalloproteinase-8 is produced primarily by chorionic cells in human fetal membranes and that the level of matrix metalloproteinase-8 protein and messenger RNA expression in fetal membranes increases during labor. Ó 2004 Elsevier Inc. All rights reserved.
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Supported by a grant from the Bill and Melinda Gates Foundation and by a grant (D43-TW-00671) from the Fogarty International Center (F.A-V.). Reprint requests: Center for Research on Reproduction and Women’s Health, University of Pennsylvania, School of Medicine, 1352 Biomedical Research Building II/III, 421 Curie Blvd, Philadelphia, PA 19104-6142. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.09.032
The primary role of the amniochorion, or fetal membranes, is to provide the fetus with a selective barrier against pathogenic organisms throughout gestation. Secondary functions include the synthesis of amniotic fluid, the maintenance of immune tolerance, and the secretion of hormones, neuropeptides, and cytokines.1 Premature rupture of the membranes (ie, rupture of the amniochorion preceding labor) often results in perinatal and neonatal infections; preterm premature rupture
844 of the membranes (ie, !37 weeks of gestation) is the leading identifiable cause of spontaneous preterm delivery.2 Unfortunately, the precise nature of the structural alterations that predispose the fetal membranes to rupture and the signals that regulate the onset of such changes remains poorly characterized. The activation of collagenolytic enzymes has been postulated to be a labor-associated event that leads to the degradation of amniochorion connective tissue and weakening of the membranes. An active connective tissue-degrading system has been demonstrated in fetal membranes under physiologic conditions, and the activity of these enzymes has been correlated with labor.3-5 Studies of proteolytic activities in fetal membranes and amniotic fluid revealed that, under normal conditions, a complex network of matrix metalloproteinases (MMPs) and their inhibitors6,7 controls the catabolism of extracellular matrix components. Several MMPs, which include MMP-2, MMP-3, and MMP-9, have been identified in human amniochorion.6,8 However, the predominant collagen in fetal membranes that is responsible for their tensile strength is collagen type I, which is degraded by the collagenases MMP-1 and MMP-8.9 The expression of MMP-1, which is produced by numerous cell types, is increased in the fetal membranes before labor,10 and levels of MMP-8 are increased in amniotic fluid of women with spontaneous term labor7,11 and in cases of intra-amniotic infection.7,12 Although MMP-8 was thought previously to be expressed exclusively by neutrophils, recent evidence has suggested that different cell types produce this MMP, including chondrocytes, fibroblasts, bronchial epithelial cells, and endothelial cells.13-15 Because the significance of MMP-8 in fetal membrane rupture is unknown, we sought to investigate the expression of MMP-8 by human fetal membranes before and after labor. The results from this study indicate that both MMP-8 protein and messenger RNA (mRNA) are produced by chorionic cells and that the increased production of MMP-8 by these cells may be important in fetal membrane rupture and labor.
Material and methods Biologic samples Fetal membranes were obtained from 2 groups of women: pregnant women (n = 7) with uncomplicated spontaneous labor at term and women (n = 7) with obstetric indications for elective cesarean delivery at term, without labor, and no associated pregnancy complications. No significant differences were observed in the gestational ages between both groups. Infection was excluded by clinical parameters. Tissue that was obtained from the midzone of the fetal membranes was processed immediately after deliv-
Arechavaleta-Velasco et al ery. The fetal membranes were dissected free of the placenta and were washed of all blood with the use of sterile heparinized saline solution. Adherent decidual tissue was removed with sterile cotton gauze according to the method described by Fortunato et al.16 For immunohistochemistry and in situ hybridization, samples were fixed in 10% buffered formalin overnight, washed in 50 mmol/L Tris buffer pH 7.5 that contained 0.15 mol/ L NaCl, and embedded in paraffin wax.
Preparation of amnion and chorion extracts After the maternal surface of the chorion was scraped and washed with heparinized saline solution to remove any decidual cells or blood, the fetal membranes were identified and separated manually into amnion and chorion. One to 2 g of amniochorion, amnion, or chorion were homogenized with a Polytron (Brinkmann, Westbury, NY) in 2 volumes of 50 mmol/L Tris buffer pH 7.4 that contained 0.15 mol/L NaCl, Triton X-100, and 100 mmol/L CaCl2, according to the method of Woessner and Taplin.17 Protein concentration was measured according to Bradford’s method.18
MMP-8 assay MMP-8 protein levels in amniochorion, amnion, and chorion extracts were determined with the BioTrak MMP-8 (human MMP-8, neutrophil collagenase) enzyme-linked immunosorbent assay (ELISA) system (Amersham Pharmacia Biotech, Piscataway, NJ), which is based on a 1-step sandwich enzyme-linked immunosorbent assay, according to the manufacturer’s protocol. Briefly, a 100 mL volume of the standard or the samples was transferred to a 96-well microtiter plate that had been coated previously with mouse anti-human MMP8 monoclonal antibody. The plate was incubated for 2 hours at room temperature. After being washed 3 times, the wells were incubated for 1 hour at room temperature with 100 mL of an anti-human MMP-8 polyclonal antibody that was conjugated with peroxidase. The wells were washed again 3 times; 100 mL of tetramethylbenzidine substrate solution was added, and the reaction was allowed to proceed for 30 minutes. The resulting color intensity was measured at 450 nm in an ELISA reader. Each sample was analyzed 3 times.
Western blots Western blotting was performed to confirm ELISA results and to determine whether precursor or active MMP-8 was present in the fetal membranes. Twenty micrograms of protein from amniochorion, amnion, or chorion extracts were separated by 8% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). Primary monoclonal antibody against MMP-8 (R&D Systems, Minneapolis,
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Arechavaleta-Velasco et al Table I
MMP-8 and GAPDH primer and probe sequences
Primer name
Sequence
RT-PCR MMP-8 forward MMP-8 reverse GAPDH forward GAPDH reverse
5#-ATG GAC CAA CAC CTC CGC AA-3# 5#-GTC AAT TGC TTG GAC GCT GC-3# 5#-CTG AGA ACG GGA AGC TTG TCA TCA A-3# 5#-GCC TGC TTC ACC ACC TTC TTG ATG TC-3#
Real-time RT-PCR MMP-8 forward MMP-8 reverse GAPDH forward GAPDH reverse
5#-GTG ACC CCA GTT TGA CAT TTG AT-3# 5#-TCT TTG TAG CTG AGG ATG CCT TC-3# 5#-CCC CTT CAT TGA CCT CAA CTA CA-3# 5#-CGC TCC TGG AAG ATG GTG AT-3#
In situ hybridization Sense Antisense
5#-GGC ATT CAG GCC ATC TAT GGA CTT TCA AGC AAC CCT ATC C-3# 5#-GGA TAG GGT TGC TTG AAA GTCCAT AGA TGG CCT GAA TGC C-3#
Minn) was used at 2 mg/mL and incubated with the blot overnight at 4(C. The primary antibody was detected with the use of SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill). MMP-8 that was purified from stimulated neutrophils (Calbiochem, San Diego, Calif) was used as a positive control.
Immunohistochemical determination of MMP-8 Paraffin-embedded tissues were deparaffinized and rehydrated with xylene and graded alcohol. Sections were blocked with 10% normal horse serum for 1 hour at room temperature. Subsequently, mouse monoclonal antibody raised against MMP-8 (R&D Systems) was applied at 25 mg/mL for 1.5 hour at 37(C, followed by detection with the Vectastain ABC-alkaline phosphatase anti-mouse kit (Vector Laboratories, Burlingame, Calif) and the Vector Red substrate kit (Vector Laboratories). Controls for immunohistochemistry experiments included the use of mouse immunoglobulin G as the primary antibody and the omission of primary antibody (secondary antibody alone).
Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from 500 mg of fresh tissue by TRIzol reagent (Invitrogen, Carlsbad, Calif). After RNase-free DNase (Invitrogen) treatment for 15 minutes at room temperature, RNA was further purified with RNeasy RNA isolation kit (QIAGEN, Valencia, Calif). Total RNA (2 mg) was reverse transcribed in a 20 mL reaction system with the superscript 1-step RT-PCR system (Invitrogen), according to the manufacturer’s protocol. Reverse-transcribed complementary DNA (2 mL) was amplified in a 25 mL PCR system that contained 200 mmol/L each deoxyribonucleoside triphosphate, 300 nmol/L of each primer, standard buffer supplemented
with 1 unit of platinum Taq DNA polymerase (Invitrogen), and 1.5 mmol/L MgCl2. After an initial denaturation step at 94(C for 4 minutes, 30 cycles of PCR were performed with denaturation at 94(C for 30 seconds, annealing at 56(C for 30 seconds, and extension at 72(C for 45 seconds. The last extension was performed at 72(C for 7 minutes. Specific primers (not recognizing MMP-1 or other MMPs) were designed on the basis of the published DNA sequence to amplify a 531ebase pair segment of the MMP-8 gene between nucleotides 674 and 1205 (Table I).19 Primers were also constructed for the constitutively expressed housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).19,20 Sterile water was included as a negative control in each PCR experiment.
Quantification of MMP-8 transcripts by real-time RT-PCR The mRNA level of MMP-8 was quantified by real-time RT-PCR in the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif). PCR was performed with SYBR Green PCR master mix (Applied Biosystems) according to the manufacturer’s instructions. Quantitative PCR primers are depicted in Table I. PCR cycles consisted of an initial denaturation step at 95(C for 10 minutes, followed by 40 cycles at 95(C for 15 seconds and 60(C for 60 seconds. PCR amplification of the housekeeping gene GAPDH was done for each sample as a control for sample loading and to allow normalization between samples. A standard curve was constructed with pCR-XL-TOPO vector (Invitrogen) that contained the same fragment that was amplified by the SYBR Green PCR master mix. The relative expression in each sample was calculated with respect to the standard calibration curve, as previously described.21,22 Each sample was analyzed twice; each
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Arechavaleta-Velasco et al 250 mg/mL yeast transfer RNA, 5 mg/mL polydeoxyadenylic acid,100 mg/mL polyadenylic acid, 0.05 pM/mL randomer oligonucleotide, and 500 mg/mL denatured and sheared salmon sperm DNA). Several washes at 37(C were carried out with SSC in constant agitation (2 ! SSC for 15 minutes, 1 ! SSC for 15 minutes, 0.5 ! SSC for 15 minutes). Digoxigenin was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments and thereafter by staining with NBT/ BCIP chromogen (Roche Molecular Biochemicals).
Statistical analysis Results are presented as mean G SEM of MMP-8 concentration or number of copies (MMP-8/1000 copies of GAPDH). Comparisons between groups were performed by Mann-Whitney rank sum test. A probability value of !.05 was considered to be the limit for statistical significance.
Results
Figure 1 Concentration of MMP-8 protein in (a) amniochorion, and (b) amnion and chorion extracts that were obtained from normal labor and cesarean delivery (C/S). MMP-8 levels were quantified by ELISA. Each bar represents the meanGSEM of 7 samples, which were measured in duplicate. Statistical analysis by Mann-Whitney rank sum test revealed that MMP-8 levels were significantly different (P!.05). The asterisk indicates a statistically significant difference.
PCR experiment included 2 nonetemplate control wells. PCR products were confirmed as single bands with gel electrophoresis.
In situ hybridization Specific sense and antisense probes for MMP-8 (not recognizing other MMPs) were designed, on the basis of the published complementary DNA sequence (Table I).19 These probes were labeled with digoxigenin-conjugated deoxyuridine triphosphate with the DIG oligonucleotide 3#-end labeling kit (Roche Molecular Biochemicals, Indianapolis, Ind). Six-micron sections of paraffin-embedded amniochorion that were obtained from normal labor were processed for in situ hybridization with the digoxigenin-labeled probes. Hybridization was performed at 57(C overnight in a humidified chamber with 30 ng of digoxigenin-labeled oligonucleotide probe in the hybridization solution (2 ! sodium saline citrate [SSC], 1 ! Denhardt’s solution, 10% dextran sulfate, 50 mmol/ L phosphate buffer pH 7.0, 50 mmol/L dithiothreitol,
The levels of MMP-8 were determined by ELISA in the amniochorion extracts that were obtained from normal labor and cesarean delivery. A wide standard deviation was observed in the total amount of protein that was extracted from the amniochorion. Therefore, ELISA results were normalized against the protein concentration. Enzyme immunoassay of MMP-8 revealed concentration differences between the 2 groups (Figure 1, a). Amniochorion extracts that were obtained from normal labor were increased significantly (n = 7 pregnancies; 198.80 G 26.37 ng/mg total protein) compared with those extracts that were obtained from elective cesarean delivery (n = 7 pregnancies; 40.84 G 6.77 ng/mg total protein; P ! .001). To determine which layer within the fetal membranes contained MMP-8 protein, extracts from the chorion and amnion were analyzed separately by ELISA. MMP-8 was detectable in chorion and amnion from elective cesarean delivery and labor. However, significantly higher concentrations of MMP-8 were detected in the chorion than the amnion (Figure 1, b). The levels of MMP-8 were increased significantly in chorion that was obtained after normal labor (n = 7 pregnancies; 116.0 G 12.33 ng/mg total protein), compared with chorion samples that were obtained after elective cesarean delivery (n = 7 pregnancies; 51.78 G 10.14 ng/mg total protein; P ! .001). There were no significant differences in MMP-8 concentrations between the amnion from normal labor (n = 7 pregnancies; 5.66 G 1.37 ng/mg total protein) and from elective cesarean delivery (n = 7 pregnancies; 3.81 G 1.13 ng/mg total protein). Western blots containing protein that was extracted from the same fetal membrane samples were performed to confirm the ELISA results. A representative
Arechavaleta-Velasco et al
Figure 2 Immunoreactive MMP-8 in human amniochorion. Extracts (a) from amnion (Am), chorion (Ch), and amniochorion (ACh) that were collected from cesarean delivery (C/S) or normal labor and (b) from PMN or amniochorion extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting with a murine monoclonal antibody against MMP-8. a, Amniochorion, amnion, and chorion proeMMP-8 at 65 kd and active MMP-8 at 50 kd. b, Purified human neutrophil MMP-8 with proeMMP-8 at 75 kd and active enzyme at 65 kd and amniochorion proenzyme and active enzyme at 65 and 50 kDa, respectively.
Western blot is shown in Figure 2, a. Bands at 65 and 50 kd, which correlated to proMMP-8 and MMP-8, respectively, were found in the amniochorion and chorion extracts, but not in the amnion extract that was obtained from normal labor and cesarean delivery. Molecular weight differences were observed in MMP-8 bands with protein extracts from polymorphonuclear neutrophils (PMNs) and amniochorion (Figure 2, b). The PMN extracts yielded bands at 85, 75, and 65 kd, which corresponded to preproMMP-8, proMMP-8, and active MMP-8, respectively.23,24 The 50-kd band in amniochorion and chorion most likely represents the nonglycosylated form of MMP-8.14,25 Even though immunoreactive proMMP-8 and MMP-8 were found in extracts of amniochorion and chorion collected before labor, the levels of the enzymes were always substantially lower than those detected after delivery. The findings of several Western blots confirmed that proMMP-8 and MMP-8 concentrations were increased in amniochorion and chorion extracts that were obtained from normal labor, compared with those samples obtained from cesarean delivery. MMP-8 mRNA expression in amniochorion, chorion, and amnion that was obtained from normal labor
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Figure 3 MMP-8 mRNA expression in human fetal membranes. Total RNA isolated from human amniochorion that was obtained from (a) cesarean delivery (C/S) and normal labor and from (b) delivered amnion (Am) or chorion (Ch) were transcribed into cDNA by reverse transcription. MMP-8 transcripts were amplified by PCR with specific MMP-8 oligonucleotides and subjected to agarose gel electrophoresis. PCR amplification of the housekeeping gene GAPDH was done for each sample as a control for sample loading. Realtime RT-PCR shows an incremental increase in MMP-8 expression in the amniochorion (c), amnion, and chorion (d) that were obtained from normal labor compared with those obtained from cesarean delivery (C/S). Each bar represents the mean G SEM of number of copies of MMP-8 per 1000 copies of GAPDH of 7 samples. With statistical analysis by MannWhitney rank sum test, MMP-8 mRNA levels were significantly different (P!.05). The asterisk indicates statistical significant difference.
and cesarean delivery was analyzed initially by RTPCR. This analysis revealed that MMP-8 mRNA was expressed in amniochorion that was obtained from normal labor and in the tissues that were collected from cesarean delivery (Figure 3, a). When amnion and chorion were separated manually, MMP-8 mRNA was expressed primarily by the chorion (Figure 3, b). To quantify the expression levels of MMP-8 mRNA, real-time RTPCR was performed with the same tissues. Amniochorion and chorion membranes that were obtained from normal labor showed a significant increase (P = .03) of MMP-8 mRNA expression, compared with those samples that were obtained from cesarean delivery (Figure 3, c and d). In contrast with the qualitative RT-PCR, the increased sensitivity of this technique permitted us to show that the amnion that was obtained from normal labor expressed low levels of the MMP-8 mRNA (Figure 3, d). No band was observed with the negative control (data not shown).
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Figure 4 Immunohistochemical localization of MMP-8 in fetal membranes. Section of fetal membranes were obtained from cesarean delivery (a: original magnification, !10; c: original magnification, !20) and normal labor (b: original magnification, !10; d: original magnification, !20). MMP-8 is located mainly in chorionic cells in fetal membranes that were obtained from normal labor (b, d). The arrows point to cells with positive staining. The arrowheads point to the amnion.
To localize MMP-8 protein in amniochorion membranes, immunohistochemistry with antieMMP-8 antibody was carried out. The expression of MMP-8 protein was observed in the chorionic layer of the membranes that were obtained from normal labor and cesarean deliveries (Figure 4). Immunoreactive protein was distributed homogeneously within the cell cytoplasm. The fetal membranes from patients with normal labor demonstrated a higher intensity of staining for this enzyme. Faint staining was observed in the amnion cells of the samples that were obtained from normal labor (Figure 4, a). No positive cells were detected in the amnion of fetal membranes that were obtained from cesarean delivery (Figure 4, b). Immunoreactive protein in the amnion was present mainly on the surface of the amnion cells. These results were consistent in the 7 samples from each group that were analyzed. Neither control (goat immunoglobulin G as the primary antibody or secondary antibody alone) resulted in any appreciable staining of the amniochorion (data not shown). To localize the exact site and cellular distribution of MMP-8 mRNA expression in amniochorion that was obtained from women after labor at term, in situ hybridization was performed. Similar to the immunohisto-
Arechavaleta-Velasco et al
Figure 5 Localization of MMP-8 transcripts in human amniochorion that was collected from normal labor by in situ hybridization histochemistry. Fetal membranes were obtained from normal labor and hybridized with the MMP-8 antisense probe (a: original magnification, !10; c: original magnification, !20). The red reaction product indicates the presence of MMP-8 mRNA in the chorionic cells. No signals were detectable in parallel sections that were hybridized with the MMP-8 sense probe (b: original magnification, !10; d: original magnification, !20).
chemistry findings, MMP-8 in situ hybridization signals with the antisense probe were located mainly in the chorionic cells of membranes that were collected after labor (Figure 5, a and c). Hybridization signals were not detected when a sense probe was used (negative control; Figure 5, b and d).
Comment MMPs have been implicated in the mechanisms that lead to membrane rupture during labor.2,6,8,26 Human collagenases (MMP-1, MMP-8, and MMP-13) share the unique ability to cleave the triple helical domain of fibrillar collagen types I, II, and III, which form the backbone of the extracellular matrix of the fetal membranes and are responsible for most of the amnion tensile strength.9,27 MMP-8 is regarded as a neutrophilspecific MMP that is stored in granules and released on cell activation.27 This study shows, for the first time, that MMP-8 is expressed by chorionic cells in the human fetal membranes. The expression of MMP-8 was demonstrated with the use of Western blotting and RT-PCR. The predominant form of active MMP-8 in amniochorion
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Arechavaleta-Velasco et al and chorion migrated on sodium dodecylsulfate-polyacrylamide gel electrophoresis at a size of 50 kd and appears to be generated from a 65-kd latent MMP-8, which is in contrast with the 65-kd active and 85-kd latent forms of MMP-8 that were detected in amniotic fluid specimens that were obtained by amniocentesis from women with intra-amniotic infection.12 Although there is a discrepancy in the sizes of active and latent MMP-8 that were obtained from amniotic fluid and fetal membrane extracts, the MMP-8 species that were observed by immunoblotting in our study are comparable to the molecular masses of latent and active MMP-8 that were reported previously in other cell types.14,25 In addition, the direct comparison of PMN and amniochorion MMP-8 shows that the molecular weights of both types of the enzyme are different. These differences may be due to the degree of protein glycosylation between the chorionic and neutrophilic MMP-8. Enzyme-linked immunosorbent assay, but not Western blot, revealed that MMP-8 was present in the amnion; this discrepancy may be due to the sensitivity between the 2 techniques. Immunocytochemical localization revealed that the MMP-8 was located mainly on the surface of amnionic cells. Real-time RT-PCR results revealed that MMP-8 mRNA was expressed at low levels in this tissue. These results raise the possibility that most of the MMP-8 that is present in the amnion might be translocated to the surface of the amnionic cells after being synthesized in the chorionic cells. A similar observation was made previously with the secretion of MMP-2 and MMP-9 in a 3-dimensional co-culture of normal endometrial stromal cells and endometrial cancer cells.28 The striking increase in MMP-8 that we observed during labor appears to be a consequence of both increased MMP-8 mRNA expression and MMP-8 protein synthesis. The sites of MMP-8 synthesis in the amniochorion were determined by immunohistochemistry and in situ hybridization. In the fetal membranes that were obtained after labor, MMP-8 protein and mRNA were co-localized in the chorionic cells. Our findings are congruent with previous reports that demonstrated that MMP-8 concentrations in amniotic fluid are elevated during spontaneous term labor.7 It is possible that neutrophils are recruited and activated in the membranes, uterine wall, and cervical stroma during parturition.29 However, our data show that the chorionic cells, in combination with neutrophils, may be responsible for the MMP-8 increment. The increase in MMP-8 in the amniochorion provides a mechanism for the catabolism of collagen type I, the main collagen of fetal membranes.9 The denatured collagen type I fragments and collagen type IV are substrates for MMP-9, which are also increased in the amniochorion during labor.8 Thus, the combined actions of MMP-8, MMP-9, and other MMPs may provide a mechanism for weakening of the fetal membranes during labor.
The collagenolytic process in fetal membranes that is associated with normal labor is due to an increase of MMPs under the influence of physiologic signals, such as platelet-activating factor, endothelins, prostaglandins, and cytokines.30-32 Although we did not study MMP-8 regulation, previous reports establish that MMP-8 can be induced in other tissues by inflammatory cytokines.13,14 Therefore, we hypothesize that, as with other members of the MMP family in the amniochorion,32 the expression of MMP-8 could be regulated by tumor necrosis factorea and interleukin-1b. Additional studies that will investigate the factors that induce MMP-8 expression in fetal membranes and the expression of MMP-8 in these tissues in cases of intra-amniotic infection are warranted. In summary, MMP-8 was considered previously to be expressed exclusively by neutrophils. The results of the present study show that chorionic cells are able to express MMP-8 mRNA and protein in vivo. The fact that chorionic cells express MMP-8 suggests that MMP-8 is one of the proteinases in amniochorion that are responsible for degrading collagen type I, which results in a progressive loss of amniochorion tissue strength that may lead to its breakdown.
Acknowledgments We thank Dr Jose R. Conejo-Garcia for his help with the real-time RT-PCR technique.
References 1. Petraglia F, Florio P, Napp C, Genazzani AR. Peptide signaling in human placenta and membranes: autocrine, paracrine and endocrine mechanisms. Endocr Rev 1996;17:156-86. 2. Parry S. Strauss JF III. Premature rupture of the fetal membranes. N Engl J Med 1998;338:663-70. 3. Halaburt JT, Uldbjerg N, Helming R, Ohlsson K. The concentration of collagen and the collagenolytic activity in the amnion and the chorion. Eur J Obstet Gynecol Reprod Biol 1989;31:75-82. 4. Bryant-Greenwood GD, Yamamoto SY. Control of peripartal collagenolysis in the human chorion-decidua. Am J Obstet Gynecol 1995;172:63-70. 5. Fortunato SJ, Menon R. Screening of novel matrix metalloproteinases (MMPs) in human fetal membranes. J Assist Reprod Genet 2002;19:483-6. 6. Fortunato SJ, Menon R, Lombardi SJ. Collagenolytic enzymes (gelatinases) and their inhibitors in human amniochorionic membrane. Am J Obstet Gynecol 1997;177:731-41. 7. Maymon E, Romero R, Pacora P, Gomez R, Athayde N, Edwin S, et al. Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, premature rupture of the membranes, and intrauterine infection. Am J Obstet Gynecol 2000;183: 94-9. 8. Vadillo-Ortega F, Gonzalez-Avila G, Furth EE, Hanqin L, Muschel R, Stetler-Stevenson W, et al. 92 kDa type IV collagenase (matrix metalloprotinase-9) activity in human amniochorion increases with labor. Am J Pathol 1995;146:148-56. 9. Bryant-Greenwood G. The extracellular matrix of the human fetal membranes: structure and function. Placenta 1998;19:1-11.
850 10. Vettraino IM, Roby J, Tolley T, Parks WC. Collagenase-I, stromelysin-I, and matrilysin are expressed within the placenta during multiple stages of human pregnancy. Placenta 1996;17:557-63. 11. Maymon E, Romero R, Chaiworapongsa T, Berman S, Conoscenti G, Gomez R, et al. Amniotic fluid matrix metalloproteinase-8 in preterm labor with intact membranes. Am J Obstet Gynecol 2001;185:1149-55. 12. Angus S, Segel S, Hsu CD, Laksmith G, Clark P, Sammel M, et al. Amniotic fluid matrix metalloproteinase-8 indicates intra-amniotic infection. Am J Obstet Gynecol 2001;185:1232-8. 13. Cole AA, Chubinskaya S, Schumacher B, Huch K, Szabo G, Yao J, et al. Chondrocyte matrix metalloproteinase-8: human articular chondrocytes express neutrophil collagenase. J Biol Chem 1996; 271:11023-6. 14. Hanemaaijer R, Sorsa T, Konttinen YT, Ding Y, Swutinen M, Visser H, et al. Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells: regulation by tumor necrosis factor-alpha and doxycycline. J Biol Chem 1997; 272:31504-9. 15. Prikk I, Maisi P, Pirila¨ E, Sepper R, Salo T, Wahlgren J, et al. In vivo collagenase-2 (MMP-8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis. J Pathol 2001;194:232-8. 16. Fortunato SJ, Menon R, Swan KF, Lyden TW. Organ culture of amniochorionic membrane in vitro. Am J Reprod Immunol 1994; 32:184-7. 17. Woessner JF, Taplin C. Purification and properties of a small latent matrix metalloproteinase of the rat uterus. J Biol Chem 1988;263:16918-25. 18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 1976;72:248-54. 19. Hasty KA, Pourmotabbed TF, Goldberg GI, Thompson JP, Spinella DG, Stevens RM, et al. Human neutrophil collagenase: a distinct gene product with homology to other matrix metalloproteinases. J Biol Chem 1990;265:11421-4. 20. Ercolani L, Florence B, Denaro M, Alexander M. Isolation and complete sequence of a functional human glyceraldehyde3-phosphate dehydrogenase gene. J Biol Chem 1988;263: 15335-15341. 21. Garcia JR, Krause A, Schulz S, Rodriguez-Jimenez FJ, Kluver E, Adermann K, et al. Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB J 2001;15:1819-21.
Arechavaleta-Velasco et al 22. Garcia JR, Jaumann F, Schulz S, Krause A, Rodriguez-Jimenez J, Forssmann U, et al. Identification of a novel, multifunctional betadefensin (human beta-defensin 3) with specific antimicrobial activity: Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res 2001;306:257-64. 23. Knauper V, Kramer S, Reinke H, Tschesche H. Characterization and activation of procollagenase from human polymorphonuclear leucocytes. N-terminal sequence determination of the proenzyme and various proteolytically activated forms. Eur J Biochem 1990; 189:295-300. 24. Knauper V, Osthues A, DeClerck YA, Langley KE, Blaser J, Tschesche H. Fragmentation of human polymorphonuclearleukocyte collagenase. Biochem J 1993;291:847-54. 25. Hasty KA, Jeffrey JJ, Hibbs MS, Welgus HG. The collagen substrate specificity of human neutrophil collagenase. J Biol Chem 1987;262:10048-52. 26. Athayde N, Edwin SS, Romero R, Gomez R, Maymon E, Pacora P, et al. A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes. Am J Obstet Gynecol 1998;179: 1248-53. 27. Jeffrey JJ. Interstitial collagenases. In: Parks WC, Mecham RP, editors. Matrix metalloproteinases. San Diego (CA): Academic Press; 1998. p. 15-42. 28. Park DW, Ryu HS, Choi DS, Park YH, Chang KH, Min CK. Localization of matrix metalloproteinases on endometrial cancer cell invasion in vitro. Gynecol Oncol 2001;82:442-9. 29. Winkler M, Fischer DC, Ruck P, Marx T, Kaiserling E, Oberpichler A, et al. Parturition at term: parallel increases in interleukin-8 and proteinase concentrations and neutrophil count in the lower uterine segment. Hum Reprod 1999;14:1096-100. 30. Qin X, Garibay-Tupas J, Chua PK, Cachola L, Bryant-Greenwood GD. An autocrine/paracrine role of human decidual relaxin. I: interstitial collagenase (matrix metalloproteinase-1) and tissue plasminogen activator. Biol Reprod 1997;56:800-11. 31. McLaren J, Taylor DJ, Bell SC. Prostaglandin E2-dependent production of latent matrix metalloproteinase-9 in cultures of human fetal membranes. Mol Hum Reprod 2000;6: 1033-1040. 32. Arechavaleta-Velasco F, Ogando D, Parry S, Vadillo-Ortega F. Production of matrix metalloproteinase-9 in lipopolysaccharidestimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system. Biol Reprod 2002;67:1952-8.