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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1445
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6569-6574
Copyright © 2005 by The Endocrine Society

Corticotropin-Releasing Hormone and Urocortin Induce Secretion of Matrix Metalloproteinase-9 (MMP-9) without Change in Tissue Inhibitors of MMP-1 by Cultured Cells from Human Placenta and Fetal Membranes

Wei Li and John R. G. Challis

Canadian Institutes for Health Research Group in Fetal and Neonatal Health and Development, Department of Physiology, Obstetrics and Gynecology, and Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Dr. Wei Li, 1 King’s College Circle, Medical Sciences Building, Room 3344, Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: weisun.li{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Matrix metalloproteinases (MMPs) are essential for human parturition due to their degrading of the extracellular matrix. CRH and urocortin (Ucn) are thought to play a central role in the mechanisms controlling human pregnancy and parturition.

Objective, Design, and Setting: The aim of this study was to assess the effects of CRH and Ucn on MMP-9 and tissue inhibitors of MMP-1 (TIMP-1) protein and/or mRNA levels in vitro. Zymography, Western blotting, real-time RT-PCR, and culture/treatments of purified sycytiotrophoblast, chorion trophoblast, and amniotic epithelial cells from human placenta and fetal membranes were performed.

Results: CRH and Ucn significantly increased MMP-9 protein secretion from cultured chorionic trophoblast, amnion epithelial, and syncytiotrophoblast cells (P < 0.01, compared with control, respectively), but there was no effect on TIMP-1 secretion and MMP-9 mRNA expression. Antalarmin (a CRH receptor type 1 antagonist) significantly blocked CRH- and Ucn-induced pro-MMP-9 secretion from three cell types (P < 0.01, compared with treatment with CRH and Ucn alone, respectively). Antisauvagine 30 (a CRH receptor type 2 antagonist) resulted in a significant reduction in CRH- and Ucn-induced secretion from chorionic trophoblast cells (P < 0.05) and syncytiotrophoblast cells (P < 0.01) compared with treatment with CRH and Ucn alone, respectively, but had no significant effect on amniotic epithelial cells.

Conclusion: Our results suggest that CRH and Ucn may play a role in the mechanisms controlling human parturition and preterm delivery not only by affecting myometrial contractility, but also by increasing local MMP activity in placenta and fetal membranes, thereby contributing to membrane rupture with the onset and progression of human labor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DEGRADATION OF THE extracellular matrix in the fetal membranes has been implicated in the rupture of fetal membranes, the process of parturition, and placental detachment from the decidua after parturition. A significant increase in the expression of several matrix metalloproteinases (MMP-1, -2, -3, and -9) in placenta and fetal membranes or amniotic fluid occurs with the onset of term and preterm parturition coincident with a significant decrease in the expression of tissue inhibitors of MMP (TIMP-1, -2, -3, and -4) (1, 2, 3, 4, 5, 6). The MMPs, of which there are more than 20 known, grouped into subfamilies on the basis of substrate specificity and homology, are zinc-dependent enzymes that degrade most extracellular matrices (ECM). This breakdown of ECM is correlated closely with the balance between MMPs and their endogenous inhibitors, TIMPs. It is suggested that the collagen-rich ECM of placental membranes can be degraded by increased MMPs or reduced activity of TIMPs, leading to rupture of fetal membranes and detachment of placenta from maternal uterus (1, 2, 3, 4, 5, 6, 7, 8). Relaxin and other factors can alter the expression of MMPs, suggesting that MMPs are regulatable enzymes (9, 10, 11).

CRH appears to be a key element in the control of human parturition. CRH is expressed by fetomaternal tissues, and the concentrations of CRH peptide and mRNA in the placenta increase with advancing gestation in parallel with an exponential increase in maternal plasma CRH concentrations (12, 13, 14, 15). Women with preterm labor or those with impending premature delivery have higher midpregnancy plasma CRH levels than those who deliver at term (15). Previous in vitro studies showed that CRH stimulated ACTH, prostaglandin F2{alpha} (PGF2{alpha}), and oxytocin output from human placental cells and greatly enhanced PGF2{alpha}- and oxytocin-mediated myometrial contractility (12, 16, 17, 18). Thus, it has been suggested that CRH is involved in the mechanisms that determine the duration of gestation and the onset of parturition (term and preterm) (19, 20, 21). Urocortin (Ucn), a CRH-related peptide, is also synthesized by fetomaternal tissues, but its plasma levels increase only after the onset of parturition (22, 23). It has similar biological effects as CRH, augmenting PGF2{alpha}-mediated myometrial contractility and ACTH and PG release from cultured human placental cells through the same CRH receptors (24).

At the present time, the mechanisms regulating MMP-9 production by human placenta and fetal membranes remain poorly understood, and there is no information relating to the possible effects of CRH and Ucn on MMP secretion from human placental and fetal membranes. Therefore, the purpose of the present study was to investigate whether CRH and Ucn could regulate MMP-2, MMP-9, and TIMP-1 production in cultured human placental and fetal membrane cells and to use different CRH receptor antagonists to determine which CRH receptor subtype might be involved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human fetal membrane collection and cell cultures

Placentas with attached fetal membranes were collected from normal term (>37 wk gestation) pregnancies after elective cesarean delivery (nonlabor; n = 16). None of the patients had received PGs, corticosteroids, or oxytocin. Patient consent and ethical approval were obtained before tissue collection in accordance with the Canadian Tri-Council guidelines and the regulations of Mount Sinai Hospital (Toronto, Canada) and the University of Toronto. Syncytiotrophoblast, chorionic trophoblast, and amniotic epithelial cells were prepared using the methods described previously (25). Cells were plated in 24-well plates (0.8–1 x 106 cells/well for zymography) or 8–10 x 106 cells in 60-mm diameter dishes (for real-time RT-PCR and Western blotting) and cultured in DMEM supplemented with 10% fetal calf serum (Invitrogen Life Technologies, Inc., Gaithersburg, MD) and antibiotics (1000 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.23 µg /ml amphotericin; Sigma-Aldrich Corp., St. Louis, MO) at 37 C under 5% CO2/95% O2 for 72 h. The serum-free DMEM was then used to replace the culture medium. After 12-h preincubation, the hormones and antagonists were added and incubated for 18 h or other time periods.

CRH, Ucn, astressin, and phorbal 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich Corp.; antisauvagine 30 was purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA); antalarmin was a gift from Dr. George Chrousos (National Institutes of Health, Bethesda, MD). Substances were used at final concentrations ranging from 10–12–10–6 M to embrace the maternal or intrauterine tissue concentrations found in human pregnancy at term. Vehicle-treated wells (controls) were present in each experiment. After incubation, the medium was harvested and stored at –20 C until MMP-9 and TIMP-1 assays. Cells were used for RNA extraction.

Zymography for MMP-2 and MMP-9

About 15–30 µl (10 µg cell protein) harvested culture medium and 30 µg/lane MMP-9/MMP-2 mixed standard (BIOMOL, Plymouth Meeting, PA) were electrophoresed under nonreducing conditions in a 10% acrylamide gel containing 1 mg/ml gelatin (Sigma-Aldrich Corp.), according to the method described by Fisher and Werb (26). After electrophoresis, the gels were washed at room temperature for 1 h in 2.5% Triton X-100 and 50 mM Tris-HCl (pH 7.5), then incubated at 37 C overnight in buffer containing 150 mM NaCl, 5 mM CaCl2, and 50 mM Tris-HCl (pH 7.6). Thereafter, gels were stained with 0.1% (wt/vol) Coomassie Brilliant Blue R-250 in 30% (vol/vol) isopropyl alcohol/10% glacial acetic acid for 60 min and destained in 10% (vol/vol) methanol/5% (vol/vol) glacial acetic acid. Semiquantification of the bands corresponding to 92-kDa gelatinase was performed by densitometry using Scion Image software (Scion Corp., Frederick, MD).

Western blotting analysis

About 70 µl harvested culture medium and 15 µg/lane MMP-9/MMP-2 mixed standard were incubated in SDS-PAGE sample buffer, subjected to SDS-PAGE analysis with 10–12% acrylamide gel, and electrotransferred onto a nitrocellulose membrane. The membrane was blocked in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 overnight. The membrane was then incubated with one of the following antibodies: mouse monoclonal antihuman MMP-9 (Oncogene Research Products, San Diego, CA), rabbit polyclonal antihuman MMP-2 (BIOMOL), and rabbit polyclonal antihuman TIMP-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was then incubated with the appropriate secondary antibodies of either peroxidase-conjugated sheep antimouse IgG or peroxidase-conjugated donkey antirabbit IgG (Amersham Biosciences, Arlington Heights, IL). Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (PerkinElmer).

RNA isolation and real-time RT-PCR analysis

Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions, and the RNA was reverse transcribed using SuperScript reverse transcriptase (Invitrogen Life Technologies, Inc.). The forward and reverse primers used were 5'-GTGCTGGGCTGCTGCTT-TGCTG-3' and 5'-GTCGCCCTCAAAGGTTTGGAAT-3' for MMP-9 and 5'-GGGGCTTCA-CCAAGACCTACAC-3' and 5'-AAGAAAGATGGGAGGGGAACA-3' for TIMP-1. Real-time PCR was performed using the Platinum qPCR Supermix-UDG Kit (Invitrogen Life Technologies) on a Rotor-Gene SG3000 system (Montreal Biotech, Inc., Montreal, Canada). PCR cycles consisted of an initial denaturation step at 95 C for 5 min, followed by 40 cycles at 95 C denaturation for 30 sec at 60 C annealing for 30 sec, and 72 C extension for 30 sec. Amplification of the housekeeping gene, ß-actin, was measured for each sample as an internal PCR control for sample loading and normalization. To determine the relative quantitation of gene expression for both target and housekeeping genes, the comparative threshold cycle (Ct) method with arithmetic formulas was used. Subtracting the Ct of housekeeping gene from the Ct of the target gene yields the {Delta}Ct in each group (control and experimental groups), which was entered into the equation 2–{Delta}Ct and calculated for the exponential amplification of PCR. mRNA levels were normalized relative to ß-actin values.

Statistical analysis was carried out by ANOVA and Tukey’s test. Results are expressed as the mean ± SEM for the number of different experiments studied. Control cultures were conducted in the absence of exogenous CRH or Ucn. Statistical significance was set at P < 0.05. Calculations were performed using SigmaStat (Jandel Scientific software, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go presents the detection and identification of MMP-9 and MMP-2 in culture medium of human chorionic trophoblast and amniotic epithelial and syncytiotrophoblast cells by zymography and Western blotting. MMP expression was detected in the culture medium of human chorionic trophoblasts by zymographic analysis at 92 and 72 kDa, corresponding to pro-MMP-9 and pro-MMP-2. Active MMP-9 or active MMP-2 was undetectable in all culture media under the conditions of our experiments. Only pro-MMP-9 was detected in the culture medium of amniotic epithelial and syncytiotrophoblast cells. Pro-MMP-2 was undetectable in these media under the conditions of our experiments (Fig. 1AGo). The specificity of MMP-9 and MMP-2 was confirmed by Western blot analysis using specific anti-MMP-9 and -MMP-2 antibodies (Fig. 1BGo).



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  		FIG. 1. Detection and identification of MMP-9 and MMP-2 in culture medium by human placental and fetal membrane cells. A, Representative zymograms of gelatinolytic activities in conditioned medium. M, MMP-2, MMP-9 standard; ST, syncytiotrophoblast; CT, chorion trophoblast; AE, amniotic epithelial cell. B, Western blot analysis of culture medium by chorion trophoblast cells using anti-MMP-9 and anti-MMP-2 antibodies.

 
Figure 2Go illustrates the effects of CRH and Ucn on the secretion of MMPs from cultured human placental and fetal membrane cells by zymographic analysis. The results show that CRH (10–9–10–6 M) significantly increased pro-MMP-9 secretion from chorionic trophoblasts and amniotic epithelial and syncytiotrophoblast cells at 18 h compared with controls (Fig. 2Go, panels 1, 3, and 5). Ucn also significantly stimulated output of pro-MMP-9 from these cells with concentrations of 10–9–10–8 M compared with control (Fig. 2Go, panels 2, 4, and 6). However, the level of pro-MMP-2 was not significantly changed in the medium of chorionic trophoblast cells by either CRH or Ucn (Fig. 2Go, panels 7 and 8).



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  		FIG. 2. Effects of CRH (panels 1, 3, and 5) and Ucn (panels 2, 4, and 6) on MMP-9 and MMP-2 (panels 7 and 8) secretion by chorion trophoblast (CT), amniotic epithelial (AE), and placental syncytiotrophoblast (PL). Each bar represents the relative OD (ROD) of MMP-9 or MMP-2 as a percentage of the control with representative zymography below. Data are presented as the mean ± SD from six separate experiments. *, P < 0.05; **, P < 0.01 (compared with control).

 
Figure 3Go illustrates the effect of CRH receptor antagonist on CRH- and Ucn-induced MMP-9 secretion from cultured placental and fetal membrane cells. In initial experiments with chorion trophoblast cells, we found that the stimulatory effect of CRH or Ucn on MMP-9 output was abolished in the present of the nonspecific antagonist, astressin (10–6 M), and similar results were obtained in amniotic epithelial and syncytiotrophoblast cells (Fig. 3AGo). Therefore, to investigate which CRH receptor subtype was involved in the action of CRH and Ucn on MMP-9 secretion, antalarmin, a relative specific CRH receptor type 1 antagonist, and antisauvagine 30, a more specific CRH receptor type 2 antagonist, were used. Antalarmin resulted in a significant reduction in CRH- and Ucn-induced pro-MMP-9 secretion from three cell types (Fig. 3Go, B–D). Antisauvagine 30 resulted in a significant reduction in the CRH- and Ucn-induced secretion from chorionic trophoblast and syncytiotrophoblast cells compared with treatment with CRH and Ucn alone (Fig. 3Go, B and D). However, there was no significant effect of antisauvagine 30 on MMP-9 output after CRH or Ucn on amniotic epithelial cells (Fig. 3CGo), and all antagonists alone did not change the levels of MMP-9 in culture medium (Fig. 3Go, B–D).



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  		FIG. 3. Effects of CRH and Ucn on MMP-9 secretion by chorion trophoblast (CT), amniotic epithelium (AE), and syncytiotrophoblast (PL) in the absence or presence of specific CRH receptor antagonists. For each cell type, the graphic panels represent relative OD (ROD) of MMP-9 as a percentage of the control, and the picture panels represent zymography. Data are presented as the mean ± SD. Ast, Astressin; anta, antalarmin; anti SV-30, antisauvagine 30. b, Significantly different from a and c (P < 0.05). c, Significantly different from a (P < 0.01).

 
Figure 4Go shows the change in TIMP-1, MMP-9, and MMP-2 protein secretion with CRH and Ucn by Western blot analysis. CRH or Ucn (10–8 M) had no significant effect on the secretion of TIMP-1 from chorionic trophoblast (Fig. 4Go, A1, A2, and B) or syncytiotrophoblast cells (Fig. 4Go, A3, A4, and C), although the secretion of TIMP-1 from syncytiotrophoblast cells did increase over time (Fig. 4Go, A3 and A4), compared with controls at the same time point. CRH and Ucn enhanced the secretion of MMP-9 from those cell types (Fig. 4Go, A5–A8, B, and C), but were without effect on the output of MMP-2 by chorionic trophoblast cells compared with controls at the same time point (Fig. 4Go, A9 and A10). Syncytiotrophoblast did not secrete MMP-2 (Fig. 1Go).



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  		FIG. 4. Secretion of TIMP-1 (panels 1–4), MMP-9 (panels 5–8), and MMP-2 (panels 9 and 10) by chorionic trophoblast and syncytiotrophoblast cells stimulated with CRH or Ucn (10–8 M) at different times. Representative examples of Western blotting (n = 5) are shown for different experiments in each group. B (Ba, CRH; Bb, Ucn on chorionic trophoblast cells) and C (Cc, CRH; Cd, Ucn on syncytiotrophoblast cells), Each bar represents the relative OD (ROD) of TIMP-1, MMP-9, and MMP-2 as a percentage of the control for treatment time points. *, P < 0.05 for each MMP-9 group vs. TIMP-1 group.

 
Figure 5Go illustrates the change in MMP-9 mRNA level in chorionic trophoblast and syncytiotrophoblast cells by CRH or Ucn. PMA, a potent stimulator of MMP expression in other cell systems, was used as a positive control. In both chorionic trophoblast and syncytiotrophoblast cells, CRH and Ucn (10–8 M) did not significantly affect the expression of MMP-9 mRNA after 8-h treatment. However, PMA provoked a 5- to 8-fold increase in MMP-9 mRNA expression in these cells. The results obtained at 4-, 12-, and 24-h treatments were similar to those after 8-h treatment (data not shown).



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  		FIG. 5. MMP-9 mRNA levels in chorion trophoblast and syncytiotrophoblast cells stimulated with CRH, Ucn, and PMA after 8-h culture. PMA was used as a positive control. mRNA levels were normalized to ß-actin and are expressed as the fold increase vs. control. The experiments were repeated three times with similar results. a, P < 0.001, PMA treatment vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated a potential novel role for intrauterine CRH and/or Ucn, namely, stimulation of MMP-9 protein level without change in TIMP-1 in the culture medium of chorionic trophoblast, syncytiotrophoblast, and amniotic epithelial cells. CRH- or Ucn-induced yield of MMP-9 in culture medium measured by zymography (relative OD value) was similar to our findings in tissues from vaginal deliveries (3). However, MMP-9 mRNA expression was unaffected by CRH and Ucn under the conditions of our experiments, indicating that CRH and Ucn may regulate MMP-9 production at the level of posttranslational processing, including protein synthesis and secretion. To answer this question, MMP-9 expression in cell pellets with or without CRH/Ucn remains to be investigated. Our results also show that MMP-2 expression is found only in chorionic trophoblast cells, consistent with our previous study (3). However, MMP-2 output was unchanged by CRH and Ucn. It is well known that the mechanism of regulation of MMP-2 expression is via a different pathway than that of MMP-9 in other cell systems, although they have been identified with almost identical substrate specificity (27). Taking this together with our results, it suggests that the regulation of MMP-2 production is different from that of MMP-9 in human placenta and fetal membranes as well. Although CRH and Ucn induced the secretion of MMP-9 in nonlabor placental tissue, experiments on placental tissues from term and preterm deliveries remain to be performed. Previous studies have demonstrated that MMP-1 and MMP-3 are also expressed in human placenta and fetal membranes and are increased during parturition (2, 4), but it is not known whether CRH and Ucn regulate MMP-1 and MMP-3 production.

CRH- and Ucn-induced MMP-9 secretion from chorionic trophoblast, amniotic epithelial, and syncytiotrophoblast cells was blocked by CRH receptor antagonists, indicating that CRH and Ucn regulate MMP-9 secretion through CRH receptors. This result is consistent with the report of human Ucn binding with high affinity to CRH receptors (28). Two distinct CRF receptors (CRF-R1 and CRF-R2) have been characterized: CRF-R1 consists of four isoforms (CRF-R1{alpha}, CRF-R1ß, R1C, and R1D), whereas CRF-R2 has at least three different splice variants (CRF-R2{alpha}, CRF-R2ß, and CRF-R2{gamma}). Florio et al. (29) reported that only CRF-R1{alpha} mRNA and CRF-R2 ß mRNA were present in human placental and fetal membranes. CRF-R1 mRNA was localized to the syncytiotrophoblast layer, chorionic trophoblast, amnion, and decidua, whereas CRF-R2 was mainly localized in the syncytiotrophoblast, chorionic trophoblast, and decidual cells, but was barely detectable in the amniotic epithelium. Although the splice variants CRH-R1C and R1D are expressed in placenta and fetal membranes, their expression levels are very low, and their physiological roles in these tissues are uncertain (30, 31). Our studies showed that more specific CRH receptor antagonists, antalarmin (R1 antagonist) and antisauvagine 30 (R2 antagonist), significantly blocked CRH- or Ucn-induced MMP-9 release from chorionic trophoblast and syncytiotrophoblast cells. However, only antalarmin could block the effect of CRH- or Ucn-induced MMP-9 release from amniotic epithelial cells. Our results, together with those of Florio et al. (29), might indicate that CRH and Ucn stimulate MMP-9 secretion through R1{alpha} and R2ß receptors in human chorionic trophoblast cells, but mainly through R1{alpha} receptor in human amniotic epithelial cells. Furthermore, our results showing that Ucn-induced MMP-9 secretion from amniotic epithelial cells does not occur via the R2ß receptor imply that the R1{alpha} receptor may be the major subtype by which CRH and Ucn augment MMP-9 secretion from fetal membranes. That CRH receptor antagonists did not change the release of MMP-9 by cultured cells from human placenta and fetal membranes suggests that CRH and Ucn are unlikely to be involved in basal expression of MMP-9. Our evidence, combined with the demonstrated expression of CRH and Ucn by fetomaternal tissues (13, 22) and of CRH receptor subtypes in these tissues (29), leads us to suggest a novel paracrine/autocrine role for CRH and its related peptides in the regulation of MMP secretion in vivo. The intracellular signaling pathways remain to be evaluated.

Most studies of MMPs have emphasized their key role in the breakdown of ECM that ultimately leads to the rupture of fetal membranes and detachment of the placenta from maternal uterus in human parturition (1, 2, 3, 4, 5). Simultaneously, it is well known that the activity of MMPs is restrained by TIMPs through the formation of a 1:1 complex with MMPs by which a functional balance is maintained between MMPs and TIMPs (6). It is believed that alteration of this balance represents a common pathway by which different regulators control MMP activity. Among all TIMPs, TIMP-1 is the major one that inhibits the activity of MMP-9 (5, 6). In the present study, TIMP-1 secretion was unaffected by CRH and Ucn, in contrast to their effects on MMP-9. This observation suggests that CRH and Ucn could cause an imbalance between MMP-9 and TIMP-1 expression, shifting the ratio of enzyme to inhibitor, leading to tissue degradation in the human placenta and fetal membranes.

We (19, 21) and others (32) have suggested that in women, birth results from a series of positive feedback cascades effected in an autocrine and/or paracrine manner in the fetal membranes, decidua and placenta. CRH acting via its receptors and through interactions with estrogen, glucocorticoids, prostaglandins, and oxytocin helps establish the positive feedback loops that make the smooth transition from a state of myometrial quiescence to one of contractility. However, the mechanisms leading to fetal membrane rupture are largely unknown. The degradation or remodeling of ECM macromolecules by proteolytic enzymes is a key step in the rupture of fetal membranes. MMPs are particularly implicated in this process because of their specific spectrum of substrates and their increased levels in human placenta, fetal membranes, and amniotic fluid in association with term or preterm labor. Because CRH has a central role in the control of parturition, we had considered that CRH and the related peptide, Ucn, might regulate the production of MMPs in human fetal membranes. Our results demonstrate that CRH and Ucn significantly increase MMP-9 secretion from cultured human chorionic trophoblast, amniotic epithelial, and syncytiotrophoblast cells via CRH receptors (type 1 and/or type 2) and alter the MMP-9:TIMP ratio. We speculate that CRH and Ucn may help effect synchronous regulation of myometrial contractility and degradation of ECM (at least in part), which are required for the birth process.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research.

First Published Online September 20, 2005

Abbreviations: Ct, Threshold cycle; ECM, extracellular matrix; MMP, matrix metalloproteinase; PGF2{alpha}, prostaglandin F2{alpha}; PMA, phorbal 12-myristate 13-acetate; TIMP, tissue inhibitor of matrix metalloproteinase; Ucn, urocortin.

Received June 30, 2005.

Accepted September 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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