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BOR - Papers in Press, published online ahead of print October 30, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.006452
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BIOLOGY OF REPRODUCTION 68, 1276–1281 (2003)
DOI: 10.1095/biolreprod.102.006452
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Differential Regulation of the Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases by Cytokines and Growth Factors in Bovine Endometrial Stromal Cells and Trophoblast Cell Line BT-1 In Vitro1

Michiko Hirataa,b, Takashi Sato2,a, Michiko Tsumagaria,b, Arata Shimadac,d, Haruo Nakanoc,d, Kazuyoshi Hashizumec, and Akira Itoa

a Department of Biochemistry, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan b Bio-oriented Technology Research Advancement Institution (BRAIN), Minato, Tokyo 105-0001, Japan c Laboratory of Reproductive Biology and Technology, National Institute of Agrobiological Sciences, Kukizaki, Ibaraki 305-8602, Japan d Japan Science and Technology Corporation, Chiyoda, Tokyo 102-0081, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Degradation and reconstitution of extracellular matrix in uterine endometrium is a crucial event for embryonic implantation and is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). In the present study, we investigated the regulation of MMP and TIMP expression in cultured bovine endometrial stromal cells (BESCs) and a bovine trophoblast cell line BT-1 (BT-1 cells). The production of proMMP-9 was induced by transforming growth factor ß (TGFß) and 12-O-tetradecanoylphorbol 13-acetate in the stromal cells. The treatment of BESCs with TGFß, insulin-like growth factor-I, and hepatocyte growth factor (HGF) resulted in a significant increase in the level of TIMP-1 in the culture medium. In addition, a significant increase of TIMP-2 production was observed in interleukin (IL)-1{alpha} and HGF-treated BESCs. However, the expression of TIMP-1 and TIMP-2 mRNA was not augmented by these factors. The treatment of BESCs with 12-O-tetradecanoylphorbol 13-acetate resulted in a significant increase in the level of TIMP-1 but a significant decrease in the level of TIMP-2 in the stromal cells. Membrane type-1 MMP mRNA expression in the stromal cells was augmented by tumor necrosis factor {alpha} (TNF{alpha}), IL-6, HGF, and 12-O-tetradecanoylphorbol 13-acetate. On the other hand, BT-1 cells constitutively produced proMMP-9 and proMMP-2, and the treatment of BT-1 cells with TNF{alpha}, HGF, and 12-O-tetradecanoylphorbol 13-acetate resulted in a significant increase in the level of proMMP-9 but not in the level of proMMP-2. The production of TIMP-1 in BT-1 cells was also augmented by IL-1{alpha}, TNF{alpha}, and HGF at the level of translation and was transcriptionally increased by 12-O-tetradecanoylphorbol 13-acetate. However, the level of TIMP-2 mRNA in BT-1 cells was not affected by any of the treatments. These results suggest that the expression of MMPs and TIMPs is differentially regulated by cytokines and growth factors and that the production of TIMP-1 and TIMP-2 may not be accompanied by changes in their mRNA expression in bovine endometrium and trophoblasts. Furthermore, as in humans and rodents, MMPs and TIMPs may contribute to the control of degradation and reconstitution of extracellular matrix in bovine endometrium during embryonic implantation and early placentation.

cytokines, growth factors, implantation, placenta, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryo implantation is a crucial event for development of the placenta and is initiated by an appropriate interaction between blastocysts and uterine endometrium. The maternal-fetal interaction stimulates proliferation and differentiation of trophoblasts and endometrial decidualization, which likely are spatially and temporally regulated by autocrine and paracrine systems [1, 2]. Various cytokines and growth factors derived from the maternal endometrium and blastocysts, such as interleukin (IL)-1, IL-6, transforming growth factor ß (TGFß), insulin-like growth factor (IGF), and tumor necrosis factor {alpha} (TNF{alpha}), have been associated with embryo development and implantation [3–5]. Moreover, an earlier study using knockout mice lacking the hepatocyte growth factor (HGF) gene has demonstrated that HGF is involved in placental growth and development [6].

Degradation and reconstitution of extracellular matrix (ECM) in uterine endometrium is a requisite event for enhancing trophoblast invasion and is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [2, 7, 8]. Endometrial cells and trophoblasts from human, mouse, and cow have been shown to produce gelatinase A/MMP-2, gelatinase B/MMP-9, and TIMP-1, TIMP-2, and TIMP-3 [9–12]. In addition, membrane type-1 MMP (MT1-MMP) has been found in the human placenta, and its distribution is similar to that of MMP-2 [13, 14]. Furthermore, the expression of MMPs and TIMPs in maternal endometrium and trophoblasts is regulated by IL-1, IL-6, TNF{alpha}, TGFß, IGF, and HGF [2, 15]. Therefore, these cytokines and growth factors may play important roles for successful embryo implantation and early placentation by controlling protease activities for ECM degradation.

Trophoblasts in humans and mice are highly invasive and penetrate into endometrial stromal tissues [2]. In contrast, trophoblasts in bovidae such as cows and goats are noninvasive, or the extent of invasion is very limited [16, 17]. Therefore, we speculate that the mechanism of embryo implantation in bovidae may differ from that in rodents and humans. So far, only a few reports have shown that MMP-2 and MMP-9 are expressed in bovine fetal membranes and maternal tissues [12, 18]. However, the regulatory mechanism of MMP and TIMP expression in bovine endometrial cells and trophoblasts remains unclear.

We recently established a bovine blastocyst-derived trophoblastic cell line BT-1 (BT-1 cells), which is characterized by its secretion of interferon-{tau} and placental lactogen and its differentiation into binucleate cells in vitro [19, 20]. To clarify the regulation of MMP and TIMP production in bovine endometrium and trophoblasts, we established in the present study the culture system of bovine endometrial stromal cells (BESCs) and then demonstrated, to our knowledge for the first time, that the production of MMPs and TIMPs was differentially regulated by cytokines and growth factors in BESCs and BT-1 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of BESCs

The uterus from a pregnant cow was obtained in a local abattoir, and the gestation time, which was estimated by the size of fetus, was approximately 4 mo. The BESCs were isolated from the maternal endometrium in intercaruncular, not caruncular, regions of the uterine tissue to avoid contamination of embryo-derived cells, which would be expected with use of the maternal endometrium in caruncular regions. Briefly, intercaruncular endometrial tissues were dissected and surgically separated from smooth muscle tissues. The endometrial tissues were washed with Dulbecco modified PBS (Invitrogen, Carlsbad, CA) and then incubated with PBS containing 0.1% bacterial collagenase (Wako Pure Chemical Co., Osaka, Japan) at 37°C for 1 h. The resultant cell suspension was filtered through a sterile metal mesh (40 mesh/inch) to remove residual tissues and centrifuged at 100 x g for 5 min. The precipitated small cells were plated on culture dishes coated with type-I collagen and cultured to confluence. The cells were incubated with PBS containing 0.02% EDTA at room temperature for 5 min and then incubated with 0.25% trypsin without EDTA for less than 3 min. Thereafter, almost all fibroblastic cells were detached from the culture dishes, whereas epithelial cells remained on the dishes. The BESCs were collected by centrifugation and cultured in Dulbecco modified Eagle medium (DMEM)/F12 (Invitrogen) supplemented with 10% fetal bovine serum (Asahi Techno Glass Co., Tokyo, Japan). We characterized BESCs as vimentin-positive and keratin/cytokeratin-negative cells by immunohistochemical staining with a monoclonal antibody to vimentin (clone V9; Dako Japan, Kyoto, Japan) and a monoclonal antibody to keratin/cytokeratin (Nichirei Co., Tokyo, Japan; data not shown). In these experiments, BESCs were used at the 17th to the 28th passage.

Cell Culture and Treatments

The confluent BESCs and BT-1 cells [19] in 60- or 100-mm culture dishes (Asahi Techno Glass) were washed once with DMEM/F12 supplemented with 0.2% lactalbumin hydrolysate (Sigma Chemical Co., St Louis, MO) and treated with the same medium in the presence or the absence of recombinant human IL-1{alpha} (1–10 ng/ml; a generous gift from Dainippon Pharmaceutical Co., Osaka, Japan), TNF{alpha} (1–10 ng/ml), TGFß (1–10 ng/ml), IGF-I (10–40 ng/ml), HGF (10–40 ng/ml; R&D Systems, Inc., Minneapolis, MN), IL-6 (10–40 ng/ml; Chemicon International, Inc., Temecula, CA), or 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nM; Sigma) for 24 h. The harvested culture medium was stored at -20°C until use.

Gelatin Zymography

The harvested culture medium (10 µl) was subjected to SDS-PAGE with 10% acrylamide gel containing gelatin (0.6 mg/ml; Difco Laboratories, Detroit, MI) under nonreducing condition. The gel was washed with a washing buffer (50 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 10 mM CaCl2, 1 µM ZnCl2, and 0.1% Triton X-100) to remove SDS and then incubated at 37°C in an incubation buffer (50 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 10 mM CaCl2, and 1 µM ZnCl2). Thereafter, the gel was stained with Coomassie brilliant blue R-250, and gelatinolytic activity was detected as unstained bands on a blue background.

Western Blot Analysis for TIMPs

The harvested culture medium (1 ml) was subjected to SDS-PAGE with 12.5% acrylamide gel under reducing condition. The proteins separated in the gel were electrotransferred onto a nitrocellulose membrane, and the membrane was reacted with mouse anti-(bovine TIMP-1) or anti-(human TIMP-2) antibody (generous gifts from Dr. K. Iwata, Daiichi Fine Chemical Co., Toyama, Japan), which was then complexed with horseradish peroxidase-conjugated goat anti-(mouse immunoglobulin [Ig] G) IgG (Sigma). Immunoreactive TIMP-1 and TIMP-2 were visualized with enhanced chemiluminescence-Western blotting detection reagents (Amersham Biosciences, Tokyo, Japan) according to the manufacturer's instructions. The relative amounts of those proteins were quantified by densitometric scanning using the Image Analyzer LAS-1000 plus (Fuji Film Co., Tokyo, Japan).

Northern Blot Analysis

Cytoplasmic RNA (20 µg) isolated from BESCs and BT-1 cells was electrophoresed on a 1.0% formaldehyde-denatured agarose gel and then transferred onto a nylon membrane. The membrane was hybridized with 32P-labeled human cDNA probes for proMMP-1, proMMP-3, and MT1-MMP [21] as well as for TIMP-1 and TIMP-2 [22]. The relative amounts of steady-state mRNA were quantified by densitometric scanning using the Image Analyzer LAS-1000 plus (Fuji Film) and calculated after correction for the level of 28S rRNA. The cDNAs of human proMMP-1, proMMP-3, and TIMP-1 were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using cytoplasmic RNA derived from IL-1{alpha}-treated human uterine cervical fibroblasts [23] and then ligated into pGEM-T Easy vectors (Promega, Madison, WI) followed by confirmation of their cDNA sequences with Thermo Sequenase II dye terminator cycle sequencing kit (Amersham Biosciences) according to the manufacturer's instructions. The primers synthesized and utilized for the amplification were as follows: for human proMMP-1, 5'-GGTGATGAAGCAGCCCAG-3' (sense: 323–340 base pairs [bp]) and 5'-CAGTAGAATGGGAGAGTC-3' (antisense: 742–759 bp; DDBJ/EMBL/GenBank database: X54925); for human proMMP-3, 5'-GTGGAAATGAAGAGTCTT-3' (sense: 38–55 bp) and 5'-AGTCACCTCTTCCCAGAC-3' (antisense: 461–478 bp; DDBJ/EMBL/GenBank database: X05232); and for human TIMP-1: 5'-GAATTCCATGGCCCCCTTT-3' (sense: 62–74 bp) and 5'-GGATCCGGGCAGGATTCAG-3' (antisense: 683–695 bp; DDBJ/EMBL/GenBank database: X03124).

Semiquantification of TIMP-1 and TIMP-2 mRNA Levels by RT-PCR

The expression of TIMP-1 and TIMP-2 mRNA in BT-1 cells was monitored by RT-PCR. Briefly, isolated RNA (2 µg) was subjected to first-strand cDNA synthesis, and then one-tenth of the generated cDNA was used for PCR amplification. The TIMP-1 primers were the same as described above. The PCR primers for human TIMP-2 were 5'-GGTACCAGATGGGCTGCGAG-3' (sense: 710–729 bp) and 5'-TTGGAGGCCTGCTTA-3' (antisense: 925–945 bp; DDBJ/EMBL/GenBank database: J05593) and, along with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [24], were synthesized and utilized for the amplification of bovine TIMP-2 and GAPDH, respectively. The amplification was performed at 92°C for 40 sec, 56°C for 40 sec, and 72°C for 60 sec after initial denaturation at 95°C for 2 min, and the PCR for TIMP-1, TIMP-2, and GAPDH was performed with 30, 35, and 30 cycles, respectively. We confirmed that the PCR products were linearly produced between 28 and 32 cycles for TIMP-1 and GAPDH and between 30 and 35 cycles for TIMP-2. The amplified PCR products of TIMP-1 (646 bp), TIMP-2 (278 bp), and GAPDH (456 bp) were analyzed with 1% agarose gel and visualized by ethidium bromide staining. We also confirmed the cDNA sequences of the amplified PCR products after cloning them into pGEM-T vectors as described above.

Statistical Analysis

A one-way ANOVA was used for statistical analysis. The Dunnett test was applied when multiple comparisons were performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of proMMP-2 and proMMP-9 Production by Cytokines and Growth Factors in BESCs

We first examined the regulation by cytokines and growth factors of proMMP-2 and proMMP-9 production, which refers to the level of protein expression, in BESCs. As shown in Figure 1A and Table 1, TPA, which is well known as a potent inducer of MMPs and TIMPs [8, 21], caused a significant increase of 95-kDa gelatinase, corresponding to proMMP-9, in BESCs, whereas the production of proMMP-9 was not detected in the untreated cells. The treatment of BESCs with TGFß (10 ng/ml) resulted in a significant increase in the level of proMMP-9, and the augmentation was observed in a dose-dependent manner (1–10 ng/ml; data not shown). Furthermore, BESCs constitutively secreted 72-kDa gelatinase, corresponding to proMMP-2, and its production was not influenced by either the cytokines or the growth factors tested (Fig. 1B and Table 1).



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  		FIG. 1. Expression of proMMP-2, proMMP-9, and MT1-MMP in bovine endometrial stromal cells. A and B) Confluent BESCs at the 17th passage in 60-mm culture dishes were treated with IL-1{alpha} (10 ng/ml), TNF{alpha} (10 ng/ml), TGFß (10 ng/ml), IL-6 (20 ng/ml), IGF-I (20 ng/ml), HGF (20 ng/ml), and TPA (10 nM) for 24 h or were left untreated, and then the harvested culture media were subjected to gelatin zymography as described in Materials and Methods. C and D) Confluent BESCs at the 17th passage in 100-mm culture dishes were treated as described above, and then cytoplasmic RNA (20 µg) isolated from the cells was subjected to Northern blot analysis as described in Materials and Methods. Three independent experiments were highly reproducible, and typical data are shown. A) proMMP-9. B) proMMP-2. C) MT1-MMP. D) 28S rRNA. Lane 1: untreated cells; lane 2: IL-1{alpha}; lane 3: TNF{alpha}; lane 4: TGFß; lane 5: IL-6; lane 6: IGF-I; lane 7: HGF; lane 8: TPA


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  		TABLE 1. Regulation of proMMP-2, proMMP-9, and MT1-MMP expression in bovine endometrial stromal cells and BT-1 cells

We further investigated the effect of cytokines and growth factors on the mRNA expression of MT1-MMP, MMP-1, and MMP-3 in BESCs. As shown in Figure 1C and Table 1, BESCs were found to constitutively express MT1-MMP mRNA, and its level was augmented by TNF{alpha} (10 ng/ml; 1.5-fold), IL-6 (20 ng/ml; 1.5-fold), HGF (1.5-fold), and TPA (1.9-fold). In addition, TPA augmented both mRNA expression of proMMP-1/interstitial procollagenase-1 and proMMP-3/prostromelysin-1 in BESCs, but their mRNA expression was not detected in BESCs treated with either the cytokines or the growth factors (data not shown).

Regulation of TIMP-1 and TIMP-2 Production by Cytokines and Growth Factors in BESCs

As shown in Figure 2, A and B, BESCs constitutively produced TIMP-1 and TIMP-2. The treatment of BESCs with TGFß (10 ng/ml), IGF-I (20 ng/ml), and HGF (20 ng/ml) resulted in a significant increase in the level of TIMP-1 in the culture medium (1.7-, 1.6-, and 1.6-fold, respectively) (Table 2). The level of TIMP-2 in the medium was also augmented by IL-1{alpha} (10 ng/ml; 2.1-fold) and HGF (20 ng/ml; 2.6-fold) (Table 2). The TPA (10 nM) augmented the production of TIMP-1 (1.5-fold) but, in contrast, suppressed that of TIMP-2 (84% inhibition) in BESCs (Table 2). On the other hand, expression of TIMP-1 and TIMP-2 mRNA appeared not to be changed by either the cytokines or the growth factors, whereas TPA augmented the expression of TIMP-1 mRNA (1.7-fold) and suppressed that of TIMP-2 mRNA (61% inhibition) as well as their protein levels (Fig. 2, C and D, and Table 2). These results indicate that the mRNA levels of TIMP-1 and TIMP-2 were not correlated with their protein levels in BESCs treated with cytokines and growth factors.



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  		FIG. 2. Differential regulation of production and gene expression of TIMP-1 and TIMP-2 in bovine endometrial stromal cells. Confluent BESCs at the 17th passage in 60- or 100-mm culture dishes were treated as described in Figure 1, and then the harvested culture media and the isolated RNA (20 µg) were subjected to Western blot (A and B) and Northern blot analysis (C and D) as described in Materials and Methods. Three independent experiments were highly reproducible, and typical data are shown. A) TIMP-1 protein. B) TIMP-2 protein. C) TIMP-1 mRNA. D) TIMP-2 mRNA. Lane 1: untreated cells; lane 2: IL-1{alpha} (10 ng/ml); lane 3: TNF{alpha} (10 ng/ml); lane 4: TGFß (10 ng/ml); lane 5: IL-6 (20 ng/ml); lane 6: IGF-I (20 ng/ml); lane 7: HGF (20 ng/ml); lane 8: TPA (10 nM)


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  		TABLE 2. Regulation of TIMP-1 and TIMP-2 expression in bovine endometrial stromal cells and BT-1 cells

Regulation of MMP and TIMP Production in BT-1 Cells

We next examined the effect of cytokines, growth factors, and TPA on the production of MMPs and TIMPs in bovine trophoblast BT-1 cells. As shown in Figure 3A, BT-1 cells constitutively secreted 95- and 72-kDa gelatinases, both of which were of the same molecular weight as proMMP-9 and proMMP-2, respectively, from BESCs. The treatment of BT-1 cells with TNF{alpha}, HGF, and TPA resulted in a significant increase in the level of proMMP-9 in the culture medium (2.3-, 2.4-, and 13.7-fold, respectively) (Table 1). However, the production of proMMP-2 was not influenced by any stimulus tested (Fig. 3B and Table 1).



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  		FIG. 3. Expression of proMMP-2 and proMMP-9 in BT-1 cells. Confluent BT-1 cells in 60-mm culture dishes were treated as described in Figure 1, and then the harvested culture media were subjected to gelatin zymography as described in Materials and Methods. Three independent experiments were highly reproducible, and typical data are shown. A) proMMP-9. B) proMMP-2. Lane 1: untreated cells; lane 2: IL-1{alpha} (10 ng/ml); lane 3: TNF{alpha} (10 ng/ml); lane 4: TGFß (10 ng/ml); lane 5: IL-6 (20 ng/ml); lane 6: IGF-I (20 ng/ml); lane 7: HGF (20 ng/ml); lane 8: TPA (10 nM).

The BT-1 cells constitutively produced TIMP-1, and its protein level was augmented by IL-1{alpha} (1.7-fold), TNF{alpha} (1.9-fold), and HGF (1.8-fold) (Table 2) without alteration of its mRNA level (Fig. 4C and Table 2). In addition, TPA (10 nM) increased both protein and mRNA levels of TIMP-1 in BT-1 cells (2.5- and 3.3-fold, respectively) (Fig. 4C and Table 2). On the other hand, the constitutive expression of TIMP-2 mRNA was observed in BT-1 cells, but the mRNA level was not changed by any stimulus tested (Fig. 4D and Table 2). In addition, as shown in Figure 4B, TIMP-2 protein was not detected by treating the cells with selected cytokines and growth factors. Furthermore, neither the protein nor the mRNA of proMMP-1, proMMP-3, and MT1-MMP was detected in BT-1 cells under these treatments (data not shown).



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  		FIG. 4. The production of TIMP-1 and TIMP-2 does not correlate with their gene expression in BT-1 cells. Confluent BT-1 cells in 60-mm culture dishes were treated as described in Figure 1, and then the harvested culture media and the isolated RNA (2 µg) were subjected to Western blot analysis (A and B) and semiquantitative RT-PCR (C–E), respectively, as described in Materials and Methods. Three independent experiments were highly reproducible, and typical data are shown. Lane 1: untreated cells; lane 2: IL-1{alpha} (10 ng/ml); lane 3: TNF{alpha} (10 ng/ml); lane 4: TGFß (10 ng/ml); lane 5: IL-6 (20 ng/ml); lane 6: IGF-I (20 ng/ml); lane 7: HGF (20 ng/ml); lane 8: TPA (10 nM)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In bovine uterus, embryo implantation has been observed in endometrial areas associated with the caruncular structure, although caruncular regions cannot be identified in the endometrium of nonpregnant cows [25]. In addition, if BESCs are prepared from caruncular regions of the endometrium in pregnant cows, embryo-derived cells may contaminate the maternal stromal cells. Based on these structural problems, we established in the present study a culture of BESCs from the maternal endometrium in intercaruncular regions of the uterine tissue of pregnant cows. Furthermore, we assumed that BESCs in both caruncular and intercaruncular regions could be similarly affected by autocrine and paracrine factors during implantation and placentation [2, 4, 5]. Therefore, we suggest that this culture system using BESCs from intercaruncular regions may be useful in examining in vivo cellular functions of the bovine endometrium during implantation.

Degradation and reconstitution of ECM in maternal uterine endometrium is a crucial event for embryonic implantation and placentation [1, 2]. Various cytokines and growth factors are temporally expressed in maternal endometrium and cytotrophoblasts and are associated with the control of MMP-mediated ECM remodeling in humans and rodents [1–7]. So far, the expression of MMPs and TIMPs in bovine endometrium and trophoblasts has been reported in only a few papers, which indicate that proMMP-2 and proMMP-9 are produced in bovine fetal membrane and maternal tissues [12, 18]. In the present study, we demonstrated that both proMMP-2 and proMMP-9 were constitutively secreted from BT-1 cells, whereas only proMMP-2 was likewise secreted from BESCs. In addition, although proMMP-2 production was not influenced by the cytokines and growth factors tested, the production of proMMP-9 was augmented by TGFß in BESCs and by TNF{alpha} and HGF in BT-1 cells. Moreover, in human uterine fibroblasts [26–28] and cytotrophoblastic cells [29, 30], the production and activation of proMMP-9 is augmented by IL-1, TNF{alpha}, and IL-6. However, TGFß shows no effect on the expression of proMMP-9 in human endometrial stromal cells [28], whereas it increases that of trophoblastic proMMP-9 [31]. These results suggest that the production of proMMP-9 in bovine maternal endometrium and trophoblasts is differently regulated by cytokines and growth factors during implantation and placental formation. Furthermore, it seems likely that the regulation by cytokines of MMP-9 production is different in humans and cows.

The MT1-MMP has been expressed in cytotrophoblasts in early human placenta [13, 14] and mouse blastocysts [32]. Uekita et al. [33] also reported that MT1-MMP mRNA was detected in both trophoblasts and epithelial cells of endometrium from caprine placenta. In the present study, we demonstrated, to our knowledge for the first time, that MT1-MMP mRNA was expressed in BESCs and that its mRNA expression was augmented by TNF{alpha}, HGF, and TPA, as in human glioma cells [34], human fibrosarcoma HT-1080 cells [35], and human dermal fibroblasts [36]. In addition, the expression of MT1-MMP mRNA in BESCs was found to be augmented by IL-6, which is expressed in human endometrium, cytotrophoblastic cells, and syncytiotrophoblasts and may be involved in embryonic implantation and endometrial decidualization [37, 38]. Furthermore, IL-6 has been reported to enhance tumor invasion in ovarian cancer [39] and head and neck cancers [40], in which ECM degradation probably progresses because of an increase in MT1-MMP expression. Thus, these results suggest that MT1-MMP in bovine endometrium may be involved in ECM degradation during trophoblastic invasion and placental formation and that IL-6 may closely participate in bovine implantation.

The expression of MMP-1 and MMP-3 is transcriptionally induced by IL-1 and TNF{alpha} in human fibroblasts from endometrium and cervix [23, 26, 27]. In BESCs, although only TPA induced the expression of proMMP-1 and proMMP-3 mRNA, neither the cytokines nor the growth factors tested induced their mRNA expression (data not shown). In this regard, we speculate that the expression of MMP-1 and MMP-3 in BESCs may be less sensitive to these factors or may require additive stimuli.

We demonstrated that in BESCs, the production of TIMP-1 was augmented by TGFß, IGF-I, and HGF, whereas that of TIMP-2 was increased by IL-1{alpha} and HGF. In BT-1 cells, the production of TIMP-1 was also augmented by IL-1{alpha}, TNF{alpha}, and HGF, whereas the level of TIMP-2 in the culture medium was not detected. Huang et al. [28] reported that TGFß augments TIMP expression but that IL-1ß suppresses the expression of TIMP-1 and TIMP-3 mRNA in human endometrial stromal cells. In addition, a recent report by Hui et al. [41] has shown no effect of IGF-I on the expression of TIMP-1 mRNA in bovine chondrocytes. Furthermore, we found that the regulation of TIMP-1 and TIMP-2 mRNA expression by these factors did not correlate with the protein levels in both BESCs and BT-1 cells. Therefore, these results suggest that distinct and unique mechanisms may exist in the regulation of TIMP expression at the bovine fetal-maternal interface. Moreover, taken together with our previous work concerning translational regulation of TIMP-1 production [42], the regulation of TIMP production by cytokines and growth factors in bovine endometrium and trophoblasts is likely to occur at the level of translation.

The MMPs have been extracellularly or intracellularly converted to low-molecular-weight species by proteolytic deletion of the N-terminal propeptide to obtain enzymic activity [8]. In addition, ECM degradation by MMP activity is counterbalanced by the TIMPs that form a 1:1 stoichiometric complex with MMPs [43], suggesting that a balance in the ratio of MMPs:TIMPs may be important in regulating ECM remodeling of the endometrium during implantation. In the present study, we demonstrated the differential regulation of MMP and TIMP production in BESCs and BT-1 cells, but it remains unclear whether the level of MMPs overcomes that of TIMPs. Therefore, further experiments will be required to quantify the molar ratio of MMPs to TIMPs and to detect the apparent enzymic activity of MMPs.

In conclusion, we demonstrated that different sets of MMPs and TIMPs were produced in bovine endometrial stromal cells and trophoblasts and that these productions were differentially regulated by endometrial and trophoblastic cytokines and growth factors. Thus, these results suggest that, as in the case of humans and rodents, MMPs and TIMPs may contribute to the control of degradation and reconstitution of ECM in bovine endometrium during embryonic implantation and early placentation.


   ACKNOWLEDGMENTS
 
We thank Dr. K. Iwata for providing mouse anti-(bovine TIMP-1) and anti-(human TIMP-2) antibodies and Ms. A. Iwasaki for technical assistance.


   FOOTNOTES
 
1 Supported by Bio-oriented Technology Research Advancement Institution (BRAIN). Back

2 Correspondence: Takashi Sato, Department of Biochemistry, Tokyo University of Pharmacy and Life Science, School of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. FAX: 81 426 76 5734; satotak{at}ps.toyaku.ac.jp Back

Received: 15 April 2002.

First decision: 2 May 2002.

Accepted: 23 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cross JC, Werb Z, Fisher SJ. Implantation and the placenta: key pieces of the development puzzle. Science 1994 266:1508-1518[Abstract/Free Full Text]
  2. Bischof P, Meisser A, Campana A. Mechanisms of endometrial control of trophoblast invasion. J Reprod Fertil Suppl 2000 55:65-71[Medline]
  3. Austgulen R, Arntzen KJ, Vatten LJ, Kahn J, Sunde A. Detection of cytokines (interleukin-1, interleukin-6, transforming growth factor-ß) and soluble tumor necrosis factor receptors in embryo culture during in-vitro fertilization. Hum Reprod 1995 10:171-176[Abstract/Free Full Text]
  4. Kauma SW. Cytokines in implantation. J Reprod Fertil Suppl 2000 55:31-42[Medline]
  5. Salamonsen LA, Dimitriadis E, Robb L. Cytokines in implantation. Semin Reprod Med 2000 18:299-310[CrossRef][Medline]
  6. Uehara Y, Minowa O, Mori C, Shiota K, Kuno J, Noda T, Kitamura N. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 1995 373:702-705[CrossRef][Medline]
  7. Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS, Hawkes SP, Werb Z. Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development 1996 122:1723-1736[Abstract]
  8. Nagase H, Woessner JF Jr. Matrix metalloproteinases. J Biol Chem 1999 274:21491-21494[Free Full Text]
  9. Reponen P, Leivo I, Sahlberg C, Apte SS, Olsen BR, Thesleff I, Tryggvason K. 92-kDa type IV collagenase and TIMP-3, but not 72-kDa type IV collagenase or TIMP-1 or TIMP-2, are highly expressed during mouse embryo implantation. Dev Dyn 1995 202:388-396[Medline]
  10. Sharkey ME, Adler RR, Nieder GL, Brenner CA. Matrix metalloproteinase expression during mouse peri-implantation development. Am J Reprod Immunol 1996 36:72-80
  11. Hurskainen T, Hoyhtya M, Tuuttila A, Oikarinen A, Autio-Harmainen H. mRNA expressions of TIMP-1, -2, and -3 and 92-KD type IV collagenase in early human placenta and decidual membrane as studied by in situ hybridization. J Histochem Cytochem 1996 44:1379-1388[Abstract]
  12. Walter I, Boos A. Matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor-2 of matrix metalloproteinases (TIMP-2) in the placenta and interplacental uterine wall in normal cows and in cattle with retention of fetal membranes. Placenta 2001 22:473-483[CrossRef][Medline]
  13. Nawrocki B, Polette M, Marchand V, Maquoi E, Beorchia A, Tournier JM, Foidart JM, Birembaut P. Membrane-type matrix metalloproteinase-1 expression at the site of human placentation. Placenta 1996 17:565-572[CrossRef][Medline]
  14. Hurskainen T. Seiki M, Apte SS, Syrjakallio-Ylitalo M, Sorsa T, Oikarinen A, Autio-Harmainen H. Production of membrane-type matrix metalloproteinase-1 (MT1-MMP) in early human placenta. A possible role in placental implantation?. J Histochem Cytochem 1998 46:221-229[Abstract/Free Full Text]
  15. Kauma SW, Bae-Jump V, Walsh SW. Hepatocyte growth factor stimulates trophoblast invasion: a potential mechanism for abnormal placentation in preeclampsia. J Clin Endocrinol Metab 1999 84:4092-4096[Abstract/Free Full Text]
  16. Steven DH. Interspecies differences in the structure and function of trophoblast. In: Loke YW, Whyte A (eds.), Biology of Trophoblast. Amsterdam: Elsevier; 1983: 111–136
  17. Murasawa H, Takashima R, Yamanouchi K, Tojo H, Tachi C. Comparative analysis of HOXC-9 gene expression in murine hemochorial and caprine synepitheliochorial placentae by in situ hybridization. Anat Rec 2000 259:383-394[CrossRef][Medline]
  18. Maj JG, Kankofer M. Activity of 72-kDa and 92-kDa matrix metalloproteinases in placental tissues of cows with and without retained fetal membranes. Placenta 1997 18:683-687[CrossRef][Medline]
  19. Shimada A, Nakano H, Takahashi T, Imai K, Hashizume K. Isolation and characterization of a bovine blastocyst-derived trophoblastic cell line, BT-1: development of a culture system in the absence of feeder cell. Placenta 2001 22:652-662[CrossRef][Medline]
  20. Nakano H, Shimada A, Imai K, Takezawa T, Takahashi T, Hashizume K. Bovine trophoblastic cell differentiation on collagen substrata: formation of binucleate cells expressing placental lactogen. Cell Tissue Res 2002 307:225-235[CrossRef][Medline]
  21. Sato T, Koike L, Miyata Y, Hirata M, Mimaki Y, Sashida Y, Yano M, Ito A. Inhibition of activated protein-1 binding activity and phosphatidylinositol 3-kinase pathway by nobiletin, a polymethoxy flavonoid, results in augmentation of tissue inhibitor of metalloproteinases-1 production and suppression of production of matrix metalloproteinases-1 and -9 in human fibrosarcoma HT-1080 cells. Cancer Res 2002 62:1025-1029[Abstract/Free Full Text]
  22. Sato T, Sawaji Y, Matsui N, Sato H, Seiki M, Mori Y, Ito A. Heat shock suppresses membrane type 1-matrix metalloproteinase production and progelatinase A activation in human fibrosarcoma HT-1080 cells and thereby inhibits cellular invasion. Biochem Biophys Res Commun 1999 265:189-193[CrossRef][Medline]
  23. Ito A, Sato T, Ojima Y, Chen L-C, Nagase H, Mori Y. Calmodulin differently modulates the interleukin 1-induced biosynthesis of tissue inhibitor of metalloproteinases and matrix metalloproteinases in human uterine cervical fibroblasts. J Biol Chem 1991 266:13598-13601[Abstract/Free Full Text]
  24. Sato T, Kondo T, Fujisawa T, Seiki M, Ito A. Furin-independent pathway of membrane type 1-matrix metalloproteinase activation in rabbit dermal fibroblasts. J Biol Chem 1999 274:37280-37284[Abstract/Free Full Text]
  25. Wooding FBP, Flint APF. Placentation. In: Lamming GE (ed.), Marshall's Physiology of Reproduction, Pregnancy and Lactation. London: Chapman & Hall; 1994: 235–460
  26. Rawdanowicz TJ, Hampton AL, Nagase H, Woolley DE, Salamonsen LA. Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1{alpha} and tumor necrosis factor-{alpha}. J Clin Endocrinol Metab 1994 79:530-536[Abstract]
  27. Sato T, Ito A, Ogata Y, Nagase H, Mori Y. Tumor necrosis factor {alpha} (TNF{alpha}) induces pro-matrix metalloproteinase 9 production in human uterine cervical fibroblasts but interleukin 1{alpha} antagonizes the inductive effect of TNF{alpha}. FEBS Lett 1996 392:175-178[CrossRef][Medline]
  28. Huang HY, Wen Y, Irwin JC, Kruessel JS, Soong YK, Polan ML. Cytokine-mediated regulation of 92-kDa type IV collagenase, tissue inhibitor or metalloproteinase-1 (TIMP-1), and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells. J Clin Endocrinol Metab 1998 83:1721-1729[Abstract/Free Full Text]
  29. Meisser A, Chardonnens D, Campana A, Bischof P. Effects of tumor necrosis factor {alpha}, interleukin-1{alpha}, macrophage colony stimulating factor and transforming growth factor ß on trophoblastic metalloproteinases. Mol Human Reprod 1999 5:252-260[Abstract/Free Full Text]
  30. Meisser A, Cameo P, Islami D, Campana A, Bischof P. Effects of interleukin 6 (IL-6) on cytotrophoblastic cells. Mol Human Reprod 1999 5:1055-1058[Abstract/Free Full Text]
  31. Graham CH, Connelly I, MacDougall JR, Kerbel RS, Stetler-Stevenson WG, Lala PK. Resistance of malignant trophoblast cells to both the anti-proliferative and anti-invasive effects of transforming growth factor-ß. Exp Cell Res 1994 214:93-99[CrossRef][Medline]
  32. Tanaka SS, Togooka Y, Sato H, Seiki M, Tojo H, Tachi C. Expression and localization of membrane type matrix metalloproteinase-1 (MT1-MMP) in trophoblast cells of cultured mouse blastocysts and ectoplacental cones. Placenta 1998 19:41-48[Medline]
  33. Uekita T, Tanaka SS, Sato H, Seiki M, Tojo H, Tachi C. Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA in trophoblast and endometrial epithelial cell populations of the synepitheliochorial placenta of goats (Caprahircus). Arch Histol Cytol 2001 64:411-424[CrossRef][Medline]
  34. Hamasuna R, Kataoka H, Moriyama T, Itoh H, Seiki M, Koono M. Regulation of matrix metalloproteinase-2 (MMP-2) by hepatocyte growth factor/scatter factor (HGF/SF) in human glioma cells: HGF/SF enhances MMP-2 expression and activation accompanying up-regulation of membrane type-1 MMP. Int J Cancer 1999 82:274-281[CrossRef][Medline]
  35. Lohi J, Lehti K, Westermarck J, Kahari VM, Keski-Oja J. Regulation of membrane-type matrix metalloproteinase-1 expression by growth factors and phorbol 12-myristate 13-acetate. Eur J Biochem 1996 239:239-247[Medline]
  36. Han YP, Tuan TL, Wu H, Hughes M, Garner WL. TNF-{alpha} stimulates activation of pro-MMP-2 in human skin through NF-{kappa}B mediated induction of MT1-MMP. J Cell Sci 2001 114:131-139[Abstract]
  37. Jauniaux E, Gulbis B, Schandene L, Collette J, Hustin J. Distribution of interleukin-6 in maternal and embryonic tissues during the first trimester. Mol Hum Reprod 1996 2:239-243[Abstract/Free Full Text]
  38. Nasu K, Matsui N, Narahara H, Tanaka Y, Miyakawa I. Effects of interferon-{gamma} on cytokine production by endometrial stromal cells. Hum Reprod 1998 13:2598-2601[Abstract/Free Full Text]
  39. Obata NH, Tamakoshi K, Shibata K, Kikkawa F, Tomoda Y. Effects of interleukin-6 on in vitro cell attachment, migration and invasion of human ovarian carcinoma. Anticancer Res 1997 17:337-342[Medline]
  40. Nishino H, Miyata M, Kitamura K. The effect of interleukin-6 on enhancing the invasiveness of head and neck cancer cells in vitro. Eur Arch Otorhinolaryngol 1998 255:468-472[CrossRef][Medline]
  41. Hui W, Rowan AD, Cawston T. Insulin-like growth factor 1 blocks collagen release and down regulates matrix metalloproteinase-1,-3, -8, and -13 mRNA expression in bovine nasal cartilage stimulated with oncostain M in combination with interleukin 1{alpha}. Ann Rheum Dis 2001 60:254-261[Abstract/Free Full Text]
  42. Hosono T, Ito A, Sato T, Nagase H, Mori Y. Translational augmentation of pro-matrix metalloproteinase 3 (prostromelysin 1) and a tissue inhibitor of metalloproteinases (TIMP)-1 mRNAs by epidermal growth factor and transforming growth factor {alpha}, but not by interleukin 1{alpha} or 12-O-tetradecanoylphorbol 13-acetate in human uterine cervical fibroblasts: the possible involvement of an atypical protein kinase C. Biol Pharm Bull 1996 19:1285-1290[Medline]
  43. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: Evolution, structure and function. Biochim Biophys Acta 2000 1477:267-283[CrossRef][Medline]




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