Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

Preterm premature rupture of fetal membranes precedes 30-40% of preterm births. Activation of matrix metalloproteases (MMPs) is the one of the major causes of extracellular matrix (ECM) degradation in membrane rupture. Increased cortisol, regenerated by 11ß-hydroxysteroid dehydrogenase 1 in the amnion at parturition, is known to participate in a number of parturition-pertinent events. However, whether cortisol has a role in the regulation of MMPs in the membranes is not known. Here, we addressed this issue using human amnion tissue, the most tensile layer of the membranes. RNA-sequencing revealed that cortisol induced MMP7 expression dramatically in amnion fibroblasts, which was confirmed by real-time quantitative RT-PCR and Western blotting analysis in cortisol-treated amnion explants and fibroblasts. Measurement of collagen IV α5 chain (COL4A5), a substrate for MMP-7, showed that cortisol reduced its extracellular abundance, which was blocked by an antibody against MMP-7. Moreover, increased MMP-7 but decreased COL4A5 abundance was observed in the amnion tissue following labor-initiated spontaneous rupture of membranes. Mechanistic studies showed that cortisol increased the phosphorylation of c-Jun and the expression of c-Fos, the 2 major components of activated protein 1 (AP-1), respectively. The knocking down of c-Fos or c-Jun significantly attenuated the induction of MMP7 expression by cortisol. Chromatin immunoprecipitation assays showed that cortisol stimulated the enrichment of c-Fos and c-Jun at the AP-1 binding site in the MMP7 promoter. The data suggest that induction of MMP7 by cortisol via AP-1 may be a contributing factor to ECM degradation in membrane rupture at parturition.-Wang, L.-Y., Wang, W.-S., Wang, Y.-W., Lu, J.-W., Lu, Y., Zhang, C.-Y., Li, W.-J., Sun, K., Ying, H. Drastic induction of MMP-7 by cortisol in the human amnion: implications for membrane rupture at parturition.

The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro.

BACKGROUND: Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients. METHODS: Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin therapy on TNFα-induced MMP-9 gene expression and on basal MMP-9 activity and gene expression in primary amnion and chorion cells in vitro. RESULTS: Primary cells were harvested from 11 patients. Compared with the unstimulated control, TNFα increased MMP-9 activity (P = 0.005 versus control in primary amnion cells and P < 0.001 versus control in primary chorion cells) and MMP-9 gene expression (P = 0.030 versus control in primary amnion cells, P < 0.001 versus control in primary chorion cells). Compared with the unstimulated controls, MPA, but not P4 or 17P, reduced basal MMP-9 activity [mean difference (95% CI) -49.6 (-81.9, -17.3) units, P = 0.001] and gene expression [mean difference (95% CI) -53.4 (-105.9, -0.9) units, P = 0.045] in primary amnion cells. Compared with the stimulated control, MPA also reduced TNFα-induced MMP-9 activity [mean difference (95% CI) -69.0 (-91.8, -46.3) units, P < 0.001] and gene expression [mean difference (95% CI) -86.0 (-120.7, -51.3) units, P < 0.001] in primary amnion cells. Progestin pretreatment had no significant effect on basal or TNFα-induced MMP-9 activity and gene expression in primary chorion cells. CONCLUSIONS: The inhibitory effect of MPA on both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells demonstrate a possible mechanism by which progestins may prevent fetal membrane weakening leading to preterm premature rupture of membranes.

Altered expression of enzymes regulating the activity of endothelin-1 in the lower segment of the human amnion during labor.

The level of endothelin (ET)-1, a uterotonin, increases in amniotic fluid during labor. The known metallopeptidases include ET-converting enzyme (ECE), which converts inactive precursor to potent ET-1, and neutral endopeptidase (NEP), which inactivates ET-1. These enzymes are present in fetal membranes, and the aims of this study were to establish the protein expression of the enzymes within the amnion of human fetal membranes. Expressions were compared between amnions obtained before and after term labor using a Western blot analysis and enzyme-linked immunosorbent assay, respectively. The localization of these enzymes was determined using immunohistochemistry. The protein expression of the enzymes and output of bioactive ET-1 in human amnion epithelial cells (HAECs) and mesenchymal cells (HAMCs) were investigated with and without proinflammatory cytokines, oxytocin, and prostaglandin treatment. The effects of sphingosine-1-phosphate (S1P), a bioactive lipid, were also examined. The protein expression of ECE-1 was significantly increased (P < 0.01), whereas that of NEP was significantly decreased, followed by increased ET-1 (P < 0.01), in the amnion obtained after labor (P < 0.01). HAECs and HAMCs primarily expressed ECE-1 and NEP, respectively. The protein expression of ECE-1 was significantly induced (P < 0.01). However, the NEP levels were significantly reduced (P < 0.05) by treatment with TNFalpha and IL1beta followed by the 7.5-fold and 6.5-fold increase of ET-1 (P < 0.01), respectively, in the HAECs. ET-1 was increased 2-fold by S1P (P < 0.01). These results suggest that the altered expression of enzymes regulating the activity of ET-1 during parturition is controlled by inflammatory cytokines.

Protein kinase PKR-dependent activation of mitogen-activated protein kinases occurs through mitochondrial adapter IPS-1 and is antagonized by vaccinia virus E3L.

The p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) play important roles in the host innate immune response. The protein kinase regulated by RNA (PKR) is implicated in p38 MAPK activation in response to proinflammatory signals in mouse embryonic fibroblasts. To test the role of PKR in the activation of p38 and JNK MAPKs in human cells following viral infection, HeLa cells made stably deficient in PKR by using an RNA interference strategy were compared to cells with sufficient PKR. The phosphorylation of both p38 and JNK in cells with sufficient PKR was activated following either infection with an E3L deletion (DeltaE3L) mutant of vaccinia virus or transfection with double-stranded RNA (dsRNA) in the absence of infection with wild-type vaccinia virus. The depletion of PKR by stable knockdown impaired the phosphorylation of both p38 and JNK induced by either the DeltaE3L mutant virus or dsRNA but not that induced by tumor necrosis factor alpha. The PKR-dependent activation of MAPKs in DeltaE3L mutant-infected cells was abolished by treatment with cytosine beta-d-arabinoside. The complementation of PKR-deficient cells with the human PKR wild-type protein, but not with the PKR catalytic mutant (K296R) protein, restored p38 and JNK phosphorylation following DeltaE3L mutant virus infection. Transient small interfering RNA knockdown established that the p38 and JNK kinase activation following DeltaE3L infection was dependent upon RIG-I-like receptor signal transduction pathway components, including the mitochondrial adapter IPS-1 protein.

The involvement of human amnion in histologic chorioamnionitis is an indicator that a fetal and an intra-amniotic inflammatory response is more likely and severe: clinical implications.

OBJECTIVE: Amnionitis (inflammation of the amnion) is the final stage of extra-placental chorioamniotic inflammation. We propose that patients with "amnionitis", rather than "chorionitis" have a more advanced form of intra-uterine inflammation/infection and, thus, would have a more intense fetal and intra-amniotic inflammatory response than those without "amnionitis". STUDY DESIGN: The relationship between the presence of amnionitis, and a fetal and an intra-amniotic inflammatory response was examined in 290 singleton preterm births (

Tocilizumab inhibits interleukin-6-mediated matrix metalloproteinase-2 and -9 secretions from human amnion cells in preterm premature rupture of membranes.

BACKGROUND/AIMS: In the present study, we investigated the participation of inflammatory cytokine-induced mediated matrix metalloproteinase (MMP) expressions and inhibition of interleukin (IL)-6-induced MMP secretion in amniotic epithelial cells by tocilizumab. METHODS: To investigate the role of MMP expressions, immunohistochemical staining was performed using membranes obtained from 10 patients with preterm premature rupture of membranes (PPROM) and from 10 patients who underwent a nonlabor cesarean section. We also investigated the regulation of MMP expression by inflammatory cytokines in human amnion cells. RESULTS: Immunohistochemical staining showed a significantly higher expression of MMP-2 and -9 in PPROM. Treatment of cultured WISH and primary amniotic epithelial cells with 10(-8) or 10(-7)M IL-6 or tumor necrosis factor (TNF)-alpha clearly increased the secretion of MMP-2 and -9. Treatment with 10(-8)M TNF-alpha or IL-6 significantly increased the invasion of WISH or primary amniotic epithelial cells, respectively, compared with the control. At a low concentration of 1 microg/ml, tocilizumab (anti-human IL-6 receptor monoclonal antibody) inhibited the IL-6-induced MMP secretion. CONCLUSIONS: This paper is the 1st report of tocilizumab inhibiting IL-6-induced MMP-2 and MMP-9 secretions from human amnion cells in PPROM.

[Fetal membranes: embryological development, structure and the physiopathology of the preterm premature rupture of membranes]. / Les membranes foetales : développement embryologique, structure et physiopathologie de la rupture prématurée avant terme.

Fetal membranes development is a complex process. The amniotic and exo-celomic cavities are appearing first. The rapid growth of the amniotic cavity is leading to the disappearance of the exo-celomic cavity and the chorion is merging with the decidua. Fetal membranes consist of three layers: the amnion and the chorion, issued from fetal tissues and the decidua issued from maternal tissue. A balance between the synthesis and the degradation of membranes components is physiologic throughout the gestation. Two main mechanisms are involved in the degradation process: apoptosis in the cellular compartment and matrix metalloproteinase (MMP) in the extracellular matrix. Regulation of MMP is depending on factors increasing their expression (cytokines) and factors decreasing their activity tissue inhibitor of metalloproteinases (TIMPS). Particular conditions can induce an unbalance between synthesis and degradation leading to the weakening of the membranes. Different factors can be associated to induce this unbalance: infection, hormonal factors, default in membranes fusion, oxidative stress and mechanic factors. In fine, the spontaneous rupture of the membranes is always occurring in regard of the uterine cervix after a process started several weeks before.

Human amnion-derived mesenchymal stem cells induced osteogenesis and angiogenesis in human adipose-derived stem cells via ERK1/2 MAPK signaling pathway.

Mesenchymal stem cells (MSCs) have shown great potential in treating bone deficiency. Human adipose-derived stem cells (HASCs) are multipotent progenitor cells with multi-lineage differentiation potential. Human amnion-derived mesenchymal stem cells (HAMSCs) are capable of promoting osteogenic differentiation of MSCs. In this study, we investigated the effect of HAMSCs on HASCs by a transwell co-culture system. HAMSCs promoted proliferation, osteogenic differentiation, angiogenic potential and adiponectin (APN) secretion of HASCs. Moreover, the positive effect of HAMSCs was significantly inhibited by U0126, a highly selective inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway. These observations suggested that HAMSCs induced bone regeneration in HASCs via ERK1/2 MAPK signaling pathway. [BMB Reports 2018; 51(4): 194-199].

Downregulation of Jun kinase signaling in the amnioserosa is essential for dorsal closure of the Drosophila embryo.

BACKGROUND: During Drosophila embryogenesis, Jun kinase (JNK) signaling has been shown to play a key role in regulating the morphogenetic process of dorsal closure, which also serves as a model for epithelial sheet fusion during wound repair. During dorsal closure the JNK signaling cascade in the dorsal-most (leading edge) cells of the epidermis activates the AP-1 transcription factor comprised of DJUN and DFOS that, in turn, upregulates the expression of the dpp gene. DPP is a secreted morphogen that signals lateral epidermal cells to elongate along the dorsoventral axis. The leading edge cells contact the peripheral cells of a monolayer extraembryonic epithelium, the amnioserosa, which lies on the dorsal side of the embryo. Focal complexes are present at the dorsal-most membrane of the leading edge cells, where they contact the amnioserosa. RESULTS: We show that the JNK signaling cascade is initially active in both the amnioserosa and the leading edge of the epidermis. JNK signaling is downregulated in the amnioserosa, but not in the leading edge, prior to dorsal closure. The subcellular localization of DFOS and DJUN is responsive to JNK signaling in the amnioserosa: JNK activation results in nuclear localization of DFOS and DJUN; the downregulation of JNK signaling results in the relocalization of DFOS and DJUN to the cytoplasm. The HINDSIGHT (HNT) Zn-finger protein and the PUCKERED (PUC) JNK phosphatase are essential for downregulation of the JNK cascade in the amnioserosa. Persistent JNK activity in the amnioserosa leads to defective focal complexes in the adjacent leading edge cells and to the failure of dorsal closure. CONCLUSIONS: Focal complexes are assembled at the boundary between high and low JNK activity. In the absence of focal complexes, miscommunication between the amnioserosa and the leading edge may lead to a premature "stop" signal that halts dorsalward migration of the leading edge. Spatial and temporal regulation of the JNK signaling cascade may be a general mechanism that controls tissue remodeling during morphogenesis and wound healing.

The effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions.

OBJECTIVE: This study was aimed to explore the effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions. STUDY DESIGN: Zymography was conducted for matrix metalloproteinase (MMP) secretion. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to examine MMP2 transcripts, and the effect of progesterone on MMP2 promoter activity was determined with the use of luciferase activity. RESULTS: Progesterone decreased pro-MMP2 secretion, expression, and promoter activity in decidua before contractions began. The effect of progesterone was reversed completely by mifepristone (RU486). Progesterone failed to inhibit MMP2 expression in the amnion and chorion before contractions began. After contractions, progesterone failed to inhibit MMP2 expression in both the decidua and fetal membranes. CONCLUSION: MMP2 expression is inhibited by progesterone only in the decidua and only before contractions begin.

The proteolytic profile of prelabour ruptured amnion at term: a case-control study.

OBJECTIVES: The aim of this study was to investigate whether prelabour ruptured membranes at term display increased proteolytic activity and to determine whether regional structural alterations within membranes are reflective of regional variations in proteolytic activity. STUDY DESIGN: Multiple amnion samples were collected from 37 women with prelabour membrane rupture and 37 women whose membranes ruptured spontaneously during labour. In all cases the gestation was greater than 37 weeks. Substrate zymography was used to qualitatively assess gelatinase and serine protease involvement. General protease activity (metallo, serine, acid and sulfhydryl) was measured quantitatively by fluorescent substrate cleavage. RESULTS: Substrate zymography revealed no active gelatinases or serine proteases. Fluorescent studies of general protease activity showed no significant difference between the groups and no significant regional variation. CONCLUSIONS: Gelatinase and serine protease activity do not play a major role in the formation of a membrane rupture initiation site or in prelabour membrane rupture at term.

Mechanisms regulating prostaglandin H2 synthase-2 mRNA level in the amnion and chorion during pregnancy.

Increasing prostaglandin H(2) synthase (PGHS)-2 expression in the fetal membranes is implicated in the production of prostaglandins (PGs) that stimulate labour. We have determined the activity of the PGHS-2 gene in the amnion and chorion throughout gestation and defined the contribution of transcriptional and post-transcriptional mechanisms to the increase of PGHS-2 mRNA levels. We also measured PGHS-1 mRNA abundance to assess the participation of the two isoenzymes in fetal membrane PG-production during pregnancy. Amnion and chorion were collected from non-labouring women at 10-19 weeks (early), at 28-36 weeks (preterm) and at term (37-41 weeks). We determined PGHS-1 and -2 mRNA abundance and assessed PGHS-2 gene activity by measuring PGHS-2 heterogeneous nuclear RNA levels using real-time RT-PCR. PGHS-2 gene activity and mRNA levels were up-regulated in both tissues with advancing gestation. Path analysis demonstrated that the PGHS-2 mRNA up-regulation involved both transcriptional and post-transcriptional components. PGHS-2 mRNA abundance increased 9-11 fold between the early (10-19 weeks) and preterm (28-36 weeks) groups and remained high at term. The underlying mechanism was predominantly transcriptional in the amnion and post-transcriptional in the chorion. PGHS-1 mRNA expression precipitously decreased between early gestation and term. Thus, PGHS-2 mRNA abundance is up-regulated well in advance of term and is not a trigger for labour. There is a switch in PGHS mRNA expression during pregnancy with PGHS-1 dominating in the early period and PGHS-2 dominating at term.

In situ immunolabeling allows for detailed localization of prostaglandin synthesizing enzymes within amnion epithelium.

Detailed information regarding the subcellular distribution of proteins within amnion epithelial cells is a goal of numerous placental biologists. In this report, we describe a versatile technique for in situ immunolabeling in amnion that is as technically permissible as traditional immunolabeling of cultured cells and, when coupled with confocal laser scanning microscopy, is similarly capable of providing detailed information regarding subcellular protein distribution. Using antibodies directed against sequential enzymes of the prostaglandin E biosynthesis cascade, we compared this novel method with immunofluorescent labeling using amnion cells in primary culture and cryosections of reflected fetal membrane rolls. By several criteria, we observed morphological variation between the cells cultured in vitro and the tissue specimens. Despite general consistencies in immunostaining patterns between the cryosectioned specimens and those labeled in situ, morphological preservation was superior using the latter technique. Relative to the cryosectioned specimens, in situ immunostaining was advantageous in that it permitted improved sampling efficiency, and allowed regional variations in labeling to be observed in a more global context within the tissue. Our results demonstrate that in situ immunolabeling provides a useful adjunct or alternative to immunolabeling using membrane roll preparations.

Changes in matrix metalloproteinase (MMP)-2 and MMP-9 in the fetal amnion and chorion during gestation and at term and preterm labor.

Increased matrix metalloproteinase (MMP)-9 proteolytic activity is associated with term birth, preterm birth and premature rupture of membranes. However, most studies show no changes with MMP-2, which binds tightly to cell and matrix proteins. We hypothesized better protein extraction would reveal new MMP patterns. Human amnion and chorion were collected from 25 patients at preterm or term, extracted with 2% SDS (a high concentration), and the MMP protein levels and pro-enzyme activities were determined by Western immunoblotting and zymography. MMP-2 protein and MMP-2 and -9 pro-enzyme activities in the amnion increased significantly (p<0.05) with labor at term, and were higher than at preterm labor (p<0.05), when extracted with high SDS concentration. There were no changes in chorion MMPs under any condition. These associations suggest MMP-2 may be another regulator of membrane rupture and other labor-associated mechanisms at term parturition, and its role(s) should be examined further.

Dexamethasone fails to inhibit the induction of cytosolic phospholipase A(2) expression by interleukin-1beta in cultured primary human amnion fibroblasts.

OBJECTIVE: To investigate the interaction of dexamethasone and interleukin-1beta (IL-1beta) on the expression of cytosolic phospholipase A(2) (cPLA(2)), the enzyme catalyzing the first reaction in the formation of prostaglandins, in cultured primary human amnion fibroblasts. DESIGN AND METHODS: Human amnion fibroblasts were prepared from fetal amnion collected at term and were treated with dexamethasone with or without interleukin-1beta for 24h. Prostaglandin E(2) (PGE(2)) output and cPLA(2) expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA(2) promoter-driven luciferase reporter gene activity. RESULTS: Both dexamethasone and IL-1beta caused a significant increase in prostaglandin E(2) output, cPLA(2) mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1beta stimulated cPLA(2) promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1beta on PGE(2) output, cPLA(2) expression or cPLA(2) promoter-driven luciferase reporter gene activity in cultured human amnion fibroblasts. CONCLUSION: The above findings suggest that paradoxically dexamethasone is a stimulator for both prostaglandin synthesis and cPLA(2) expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1beta on prostaglandin synthesis and cPLA(2) expression is neither synergistic nor conventionally antagonistic.

Use of amnion and placenta in neonatal screening for canine GM1-gangliosidosis and the risk of diagnostic misclassifications.

BACKGROUND: A closed breeding colony of Shiba dogs with GM1-gangliosidosis is maintained at Hokkaido University (Sapporo, Japan). Neonatal genotyping is essential to control the breeding colony genetically as an animal model for the human disease. OBJECTIVES: The purpose of the present study was to determine the utility of amnion and placenta in the neonatal screening or diagnosis for canine GM1-gangliosidosis. METHODS: Twenty neonatal Shiba dogs of a pedigree with GM1- gangliosidosis were differentiated into 3 genotypes--normal, heterozygous, and affected dogs--by using a previously reported DNA mutation assay. Acid beta-galactosidase activity was measured in amnion and placenta and compared among the 3 genotypes. RESULTS: The level of beta-galactosidase activity in the amnion of affected dogs was negligible and <2% of the mean activity in normal dogs; there was no significant difference among the 3 genotypes. In placenta, beta-galactosidase activity was significantly different among all the genotypes; however, there was wide overlap in enzyme activity between normal and heterozygous dogs. The level of activity in affected dogs was relatively high and >10% of the mean activity in normal dogs. The DNA mutation assay gave correct information about genotype with genomic DNA extracted from amnion but ambiguous information with DNA from placenta. CONCLUSIONS: Amnion and placenta were not useful as enzyme sources in neonatal screening in canine GM1-gangliosidosis because of the risk of misdiagnosis. DNA from amnion is applicable as a template for genotyping, whereas placenta should not be used because canine placenta contains maternal cells.

Structures of the asparagine-linked oligosaccharides of an alkaline phosphatase, kasahara isozyme, purified from FL amnion cells.

Asparagine-linked oligosaccharides were quantitatively released by hydrazinolysis from an alkaline phosphatase, Kasahara isozyme, which was purified from FL amnion cells. Almost all of the oligosaccharides (98%) were acidic components, all of which can be converted to neutral oligosaccharides upon sialidase digestion. Structural analysis of the oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the alkaline phosphatase of FL cells contains sialylated mono-, bi-, tri-, and tetraantennary complex type sugar chains with the Gal beta 1----4GlcNAc beta 1---- outer chains. Some of the tetraantennary sugar chains contain a single Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- outer chain on their Man alpha 1----6 arm. Both fucosylated and nonfucosylated trimannosyl cores were found in the sugar chains. However, it is of interest that the core portion of monoantennary oligosaccharide was not fucosylated and that of the tetraantennary oligosaccharide with a tetrasaccharide outer chain was completely fucosylated.

[Identification of extracellular matrix metalloprotease 3 in the fetal membrane of the rat and its possible implication in the rupture of chorioamniotic membranes]. / Identificación de la metaloproteasa de matriz extracelular-3 en la membrana fetal de la rata y su posible implicación en la rotura de las membranas corioamnióticas.

OBJECTIVE: Human corioamniotic membranes, or their equivalent in the rat, function as selective barrier during gestation and their rupture is part of the mechanisms implied in the labor. Molecular mechanisms carried out in this process are unknown. TYPE OF STUDY: Experimental animal model. MATERIAL AND METHODS: Corioamniotic membranes (obtained at the beginning of the labor) of rats with programmed and synchronous pregnancies were analized. The coexistence and distribution of metalloproteinase of extracellular-3 matrix (estromelisine) in these tissues were determined. RESULTS: Secretion and tissue location of metalloproteinase of extracellular-3 matrix in fetal membranes were identified for the first time. Metalloproteinase of extracellular-3 matrix was immunolocated in the compact layer of the amnion and its secretion (by the membranes) was confirmed through electrophoresis, zimography and Western blot. By confocal microscopy it was verified that metalloproteinase of extracellular-3 matrix is located in the same places of that of extracellular-9 matrix. CONCLUSIONS: Rupture of corioamniotic membranes relates to the expression and local activity of the metalloproteinases of extracellular matrix. The coexistence of metalloproteinase of extracellular-3 matrix in the amnion of the rat has been identified; this element is added to the biochemical process of rupture, since metalloproteinase of extracelular-3 matrix is an activator of that of extracellular-9 matrix. It is possible that the physiological function of this enzyme is implied, of a main way, in the process of rupture of corioamniotic membranes during the childbirth.

Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

The mechanisms underlying human parturition are still not understood, yet we need this knowledge to combat preterm birth. Fetal membranes express abundant 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which converts inert cortisone to active cortisol. We examined whether cortisol regeneration in the amnion might play a role in human parturition through regulation of lysyl oxidase (LOX), a collagen cross-linking enzyme, thereby contributing to the rupture of fetal membranes. By using cultured human primary amnion fibroblasts, we demonstrated that, in addition to the induction of the key enzymes involved in prostaglandin E2 (PGE2) synthesis, cortisol stimulated 11ß-HSD1 and inhibited LOX reciprocally. These results were reproduced in human amnion tissue explants after cortisol treatment. Cortisone also inhibited LOX expression, which was abolished by the inhibition of 11ß-HSD1. Despite the inhibition of LOX by PGE2, inhibition of the PGE2 pathway failed to block the inhibition of LOX by cortisol. However, inhibition of glucocorticoid receptor and mutation of a negative glucocorticoid response element in LOX promoter abolished the inhibition of LOX by cortisol. Chromatin immunoprecipitation assay revealed that cortisol increased GR binding to the LOX promoter. Moreover, increased cortisol and 11ß-HSD1 abundance and decreased LOX abundance were observed in human amnion tissue after the labor-initiated spontaneous rupture of membranes. These data highlight a crucial role for local cortisol regeneration by 11ß-HSD1 in the down-regulation of LOX expression via glucocorticoid receptor binding to a negative glucocorticoid response element to its promoter in the amnion, which may contribute to rupture of fetal membranes at parturition.