ARTICLE |
Correspondence to: Helena Autio-Harmainen, Dept. of Pathology, U. of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The extracellular matrix proteolytic machinery is known to play a major role in trophoblast invasion, a process that shares similar features with the pathology of tumor invasion. In this study we investigated the expression of the recently described membrane-type matrix metalloproteinase-1 (MT-MMP-1; MMP-14) in early human placenta and decidual membrane to determine whether it might play a role in invasion. With in situ hybridization, the cytotrophoblasts of trophoblastic columns and the infiltrating intermediate trophoblasts in the decidual membrane were found to be the main producers of MT-MMP-1 mRNA. Gene expression was also seen in the villous double-layered trophoblastic epithelium and in the decidual cells of the decidual membrane. In endothelial and fibroblastic cells, however, the hybridization signal was either very weak or nonexistent. Immunohistochemical analysis and immunoelectron microscopy correlated well with the in situ hybridization findings. The most significant exception to this consisted of pericytes of spiral arteries, which appeared to lack MT-MMP-1 mRNA but showed intensive intracytoplasmic staining for the antigen. Our results show that MT-MMP-1 mRNA production is highly characteristic of intermediate trophoblasts, and MT-MMP-1 may have general importance in the tissue organization of early human placenta. We propose that MT-MMP-1 could be one of the key enzymes in the process of trophoblast invasion, acting alone or as a cell-surface activator of other proteinases. (J Histochem Cytochem 46:221229, 1998)
Key Words: MT-MMP-1, placenta, in situ hybridization, immunoelectron microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basement MEMBRANES (BMs) are specialized extracellular matrices (ECMs) of epithelial cells, which separate them from the underlying or surrounding connective tissue stroma. Degradation and penetration of ECMs, especially basement membranes, is critical for cancer cell invasion and metastasis (
Membrane-type matrix metalloproteinase-1 (MT-MMP-1) is a membrane-associated metalloproteinase discovered by PCT homology screening of placental cDNA library. It was isolated as a unique 3.4-kilobase (KB) cDNA fragment, which encodes a unique protein of 582 amino acids (Mr 66 K). Expression of COS-1 cells induced specific activation of proMMP-2 in vitro, and enhanced cellular invasion of the reconstituted BM (
In epithelial malignancies such as human colon, breast, head, and neck carcinomas, MT-MMP-1 mRNA synthesis by in situ hybridization was localized in stromal fibroblasts but not in cancer cells, which, however, showed the location of the antigen mostly intracytoplasmically and sometimes also on the carcinoma cell membrane (
Human placenta exhibits dramatic invasive properties in early pregnancy, when it is anchored to the uterine wall. Trophoblast invasion is precisely regulated to be confined spatially to the uterus and temporally to early pregnancy. Therefore, early human placenta can be regarded as a physiological counterpart to invasive malignant tumors. In this study we used in situ hybridization, immunohistochemistry, and immunoelectron microscopy to demonstrate that unorganized cytotrophoblasts of trophoblastic columns and invasive intermediate trophoblasts are the main producers of MT-MMP-1 mRNA and that villous trophoblastic epithelium is also capable of synthesizing MT-MMP-1 mRNA.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Five placentas were obtained from legal abortions at 810 weeks of gestation, performed for socioeconomic reasons. For in situ hybridization and immunohistochemistry, the placenta tissue was frozen in liquid isopentane. Altogether, 17 different samples from the five placenta were studied by in situ hybridization.
cRNA Probes and In Situ Hybridization
A 420-BP fragment extending from nt 218 to nt 638 of the human MT-MMP-1 cDNA sequence described originally by
In situ hybridization was performed as previously described by
For in situ hybridization, 8-µm-thick cyrosections were placed on SuperFrost Plus (MenzelGläser, Braunschweig, Germany) slides and fixed with 4% paraformaldehyde. After dehydration the sections were incubated in 0.2 M HCl (20 min) at room temperature (RT), followed by a 5-min wash DEPC-H2O). After treatments with proteinase K (BoehringerMannheim; Mannheim, Germany) (1 µg/ml, 30 min, 37C) and 0.2% glycine in PBS and washings twice in 1 x PBS for 30 sec, the slides were immersed in 4% paraformaldehyde in PBS for 20 min. The sections were then washed in 1 x PBS and acetylated twice in 0.25%0.5% acetic anhydride in 0.1 M triethanolamine, twice for 5 min. After a 5-min wash in PBS and dehydration, the sections were allowed to air-dry for 12 hr at RT before prehybridization with a hybridization mixture not including the labeled probe for about 2 hr at 50C. The hybridization mixture contained the labeled probe, 10 mM dithiotreitol (DTT), yeast tRNA (1 mg/ml) deionized formamide (50% of the volume), dextran sulfate (10% of the volume), 1 x SALTS [10 x SALTS; 3 M NaCl, 0.1 M Tris-HCl, 0.1 M NaPO4, 0.05 M EDTA, 0.02% Ficoll (w/v), 0.02% polyvinylpyrrolidone (PVP) (w/v), and bovine serum albumin (BSA) (0.2 mg/ml)]. The labeled riboprobes were diluted at a concentration of 3 x 106 cpm per 40-ml aliquot per slide. Before placing on sections, the labeled probe was denatured by boiling for 1 min and placed on ice. The hybridization was carried out at 50C overnight. The slides were then processed through the following stringency washes containing 50% formamide, 1 x SALTS, 10 mM DTT twice for 1 hr at 50C, followed by a wash in 0.5 M NaCl in 10 mM Tris-HCl, 1 mM EDTA (TE), pH 7.5, at 37C for 15 min, and treatment with RNase A (BoehringerMannheim) (40 mg/ml) in 0.5M NaCl in TE buffer for 30 min at 37C. After that, the sections were washed in 0.5 M NaCl in TE for 15 min at 37C and in 2 x SSC twice for 15 min at 45C and 1 x SSC twice for 15 min at 45C. After dehydration through alcohol series, the slides were air-dried and dipped in Kodak NTB-2 (Eastman Kodak; Rochester, NY) photographic emulsion diluted 1:1 with 1% glycerol in water. After exposure for 1014 days, the slides were developed in Kodak D19 (Eastman Kodak) developer and counterstained with hematoxylin and eosin.
Immunohistochemistry
For immunohistochemical staining, 4-µm cryosections from two placentas and paraffin sections from one placenta were stained using avidin and biotinylated horseradish peroxidase complex (Dako; Dakopatts, Glostrup, Denmark). Nonspecific binding was blocked with fetal calf serum (FCS) 1:5 in PBS. Sections were incubated with monoclonal antibody against MT-MMP-1 (
Immunoelectron Microscopy
Samples from the placental tissue were fixed with 8% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hr. They were cryoprotected by immersion in 2.3 M sucrose for 30 min and then frozen in liquid nitrogen.
Semithin cryosections were stained with toluidine blue and cytokeratin immunohistochemistry for light microscopic evaluation.
The sections were first incubated in 10% FCS with 0.02 M glycine in PBS (PBSglycine) on ice for 10 min. They were then incubated with the monoclonal antibody to MT-MMP-1 diluted optimally with 5% FCS in PBSglycine for 45 min and washed in PBSglycine (four changes in 15 min). After that, the sections were exposed with the antibody to rabbit anti-mouse IgG (Dako) diluted 1:200 with 5% FCS for 30 min and washed in PBSglycine (six changes in 15 min) before exposure to the protein Agold complex diluted 1:20 in 5% FCS in PBSglycine for 20 min. The sections were washed in PBSglycine (six changes in 15 min) and postfixed in 2.5% glutaraldehyde for 10 min, followed by brief washings in PBSglycine and distilled water (five changes in 10 min). Finally, the sections were embedded in 2% methylcellulose with 0.3% uranyl acetate on ice for 10 min, dried, and examined in a Philips 410 LS transmission electron microscope by using an acceleration voltage of 60 kV. In the control sections, the antibody was omitted and replaced with PBS.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Situ Hybridization and Immunohistochemical Findings
Cytotrophoblastic Columns.
In situ hybridization revealed a very strong signal for MT-MMP-1 mRNA in the unorganized cytotrophoblastic cells of trophoblastic columns. All the cells of the columns showed similar hybridization signal except for the multinucleated syncytial giant cells, in which there was considerably less label, often at the background level (Figure 1A and Figure 1B). Immunohistochemically, the mononuclear cytotrophoblasts of columns were all stained for the antigen. The staining reaction was mostly located on the cell membrane, but some cytotrophoblasts also showed intensive intracytoplasmic staining (Figure 1C). Multinucleated giant cells in the columns were negative, except for positive staining in the microvillous projections of the cell membrane (Figure 1C, inset).
|
Intermediate Trophoblasts. The intermediate trophoblasts of the trophoblastic shell and those infiltrating into the superficial decidual membrane were identified on the basis of positivity for cytokeratin (Figure 2A and Figure 2B) and for human placental lactogen (Figure 2C). Intermediate trophoblasts very intensively expressed MT-MMP-1 mRNA (Figure 3A and Figure 3B). These cells were especially numerous in the decidual membrane around spiral arteries. In such areas, the spiral artery wall and the adjacent decidual tissue often contained small fibrinoid necroses (Figure 3C). Intermediate trophoblasts exhibited an intensive, mostly intracytoplasmic staining reaction in immunohistochemical analysis (Figure 3D).
|
Placental Villi. A clear, although much fainter MT-MMP-1 mRNA expression than in cytotrophoblasts and intermediate trophoblasts was present in the cells of double-layered trophoblastic epithelium of villi (Figure 4A and Figure 4B). Both the syncytial and the cytotrophoblastic cell layers expressed MT-MMP-1 mRNA, but the signal in the syncytial layer was often more pronounced. Expression in villous endothelial and fibroblastic cells was either very week or nonexistent.
|
In immunohistochemical analysis, the cells of the cytotrophoblastic layer displayed strong labeling, which was located either intracytoplasmically or on the cell membrane. The syncytial cell layer showed a staining reaction mainly on the apical border, at which microvillous projections were stained (Figure 4C). The endothelial cells of villous capillaries and stromal fibroblasts showed a faint intracytoplasmic staining reaction.
Decidual Membrane. The large decidual cells of the decidual membrane clearly expressed MT-MMP-1 mRNA (Figure 5A and Figure 5B). They showed granular positive depositions concentrating close to the cell membrane in immunohistochemical analysis (Figure 5C). The epithelial cells of endometrial glands were negative for both MT-MMP-1 mRNA and the antigen. The endothelial cells and pericytes of spiral arteries showed either very weak or nonexistent MT-MMP-1 mRNA expression, but fibrocytic endometrial stromal cells apposing spiral arteries exhibited a strong signal for the MT-MMP-1 mRNA (Figure 3B). An intriguing immunohistochemical finding was the intensive intracytoplasmic staining reaction of the pericytes of spiral arteries (Figure 3D).
|
Immunoelectron Microscopic Findings
Immunoelectron microscopic results generally correlated with the immunohistochemistry, showing immunolabeling in the cytoplasm, in microvillous projections or close to the cell membrane in syncytial and cytotrophoblastic cells of villi, including multinucleated giant cells, and in large decidual cells of decidual membrane (Figure 6AE). Similar accentuation of labeling towards the cell membrane, as was evident by immunohistochemistry, cold not be detected by immunoelectron microscopy. Clear intracytoplasmic labeling was also seen in the endothelial and fibroblastic stromal cells of villi.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence obtained by Northern analysis indicated MT-MMP-1 mRNA expression by cells of human placental tissue. However, there is no previous knowledge about which cells are responsible for this production. Here we describe the MT-MMP-1 mRNA expression in cells of 810-gestational week human placentas and correlate the findings with the previously described expression patterns of other ECM proteins involved in matrix turnover in developing placenta. These include laminin-1, Types IV and XIII collagen, and MMP-2 and MMP-9 (
In the developing placenta, the unorganized cytotrophoblastic cells of trophoblastic columns form the cell reservoir for new villi and anchor the ovum to the uterine wall. The anchoring process involves invasion of cells to the decidual membrane, endometrium, and spiral arteries in early pregnancy. It was previously shown that cytotrophoblasts of columns were potent producers of various ECM proteins, such as MMP-2 and MMP-9, TIMP-1, -2, and -3, laminin, and Types IV and XIII collagen (
Studies by
MT-MMPs form a unique group of proteinases in the MMP superfamily (
We have previously shown that MMP-2 is localized either intracytoplasmically or on the cell membranes of cytotrophoblasts and ovarian carcinoma cells (
The presence of a complex proteolytic enzyme profile in first-trimester trophoblastic cells is consistent with the fact that first-trimester cells are capable of penetrating a variety of maternal cell layers and the associated BMs to gain access to spiral arteries. The large decidual cells of the decidual membrane contained a relatively week although clear signal for MT-MMP-1 mRNA. Because invasive intermediate trophoblasts were shown to express considerably higher levels of MT-MMP-1 mRNA than decidual cells, it can be concluded that MT-MMP-1 needed for invasion is produced mainly by the invasive cells themselves. A novel finding was also the intense intracytoplasmic staining of pericytes, although the mRNA signal in these cells was either very low or completely absent. It is possible that the MT-MMP-1 protein is synthesized elsewhere, either in decidual cells and/or stromal cells around spiral arteries, and then is selectively bound or taken up by pericytic cells.
Our results, taken together with the published biochemical studies on MT-MMP-1, suggest an important role for MT-MMP-1 in tissue organization of early placenta and in the invasion of cytotrophoblastic cells to decidua and spiral arteries. Because it is abundantly produced by invasive intermediate trophoblasts, MT-MMP-1 is proposed to be one of the key enzymes among MMPs in the process of trophoblast invasion during early pregnancy.
![]() |
Acknowledgments |
---|
Supported by the Cancer Society of Finland and the Cancer Society of Northern Finland.
We are grateful to Prof Karl Tryggvason for valuable comments during the preparation of this manuscript and to Dr Kazushi Iwata (Fuji Chemical Industries, Ltd.; Takaoka, Japan) for preparing the monoclonal antibodies. We also thank Ms Annikki Huhtela and Ms Riitta Karvonen for expert technical assistance and Mr Tapio Leinonen for preparing the micrographs.
Received for publication June 6, 1997; accepted August 5, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander CM, Werb Z (1992) Targeted distribution of the tissue inhibitor of metalloproteinases gene increases the invasive behaviour of primitive mesenchymal cells derived from embryonic stem cells in vitro. J Cell Biol 118:727-739[Abstract]
AutioHarmainen H, Hurskainen T, Niskasaari K, Höyhtyä M, Tryggvason K (1992) Simultaneous expression of 70 kilodalton type IV collagenase and type IV collagen 1(IV) chain genes by cells of early human placenta and gestational endometrium. Lab Invest 67:191-200[Medline]
AutioHarmainen H, Karttunen T, Hurskainen T, Höyhtyä M, Kauppila A, Tryggvason K (1993) Expression of 72 kilodalton type IV collagenase (gelatinase A) in benign and malignant ovarian tumors. Lab Invest 69:312-321[Medline]
AutioHarmainen H, Sandberg M, Pihlajaniemi T, Vuorio E (1991) Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta. Lab Invest 64:483-491[Medline]
Cao J, Sato H, Takino T, Seiki M (1995) The C-Terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. J Biol Chem 270:801-805
Docherty A, Murphy G (1990) The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins. Ann Rheum Dis 51:469-479[Abstract]
Emonard HP, Remacle AG, Noel AC, Grimaud J-A, StetlerStevenson WG, Foidart J-M (1992) Tumor cell surface-associated binding site for the Mr 72,000 type IV collagenase. Cancer Res 52:5845-5848[Abstract]
Höyhtyä M, Fridman R, Komarek D, PorterJordan K, StetlerStevenson WG, Liotta LA, Liang CM (1994) Immunohistochemical localization of matrix metalloptroteinase 2 and its inhibitor TIMP-2 in neoplastic tissues with monoclonal antibodies. Int J Cancer 56:500-505[Medline]
Hurskainen T, Höyhtyä M, Tuuttila A, Oikarinen A, AutioHarmainen H (1996) mRNA expression 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 44:1379-1387[Abstract]
Juvonen M, Pihlajaniemi T, AutioHarmainen H (1993) Location and alternative splicing of type XIII collagen RNA in the early human placenta. Lab Invest 69:541-551[Medline]
Kurman RJ, Main CS, Chen H-C (1984) Intermediate trophoblast: a distinctive form of trophoblast with specific morphological, biochemical and functional features. Placenta 5:349-369[Medline]
Librach CL, Werb Z, Fitzgerald ML, Chin K, Corwin NM, Estevas RA, Grobelny D, Galardy R, Damsky CH, Fisher SJ (1991) 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Biol Chem 113:437-449
Mignatti P, Rifkin DB (1993) Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 73:161-195
Naylor MS, Stamp GW, Davies BD, Balkwill FR (1994) Expression and activity of MMPs and their regulators in ovarian cancer. Int J Cancer 58:50-56[Medline]
Nomura H, Sato H, Seiki M, Mai M, Okada Y (1995) Expression of membrane-type matrix metalloproteinase in human gastric carcinomas. Cancer Res 55:3263-3266[Abstract]
Okada A, Bellocq J-P, Rouyer N, Chenard M-P, Rio M-C, Chambon P, Basset P (1995) Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc Natl Acad Sci USA 92:2730-2734[Abstract]
Polette M, Nawrocki B, Pintiaux A, Massenat C, Volders E, Schaaps JP, Birembaut P, Foidart JM (1994) Expression of gelatinases A and B and their tissue inhibitors by cells of early and term placenta and gestational endometrium. Lab Invest 72:838-846
Puente XS, Pendas AM, Llano E, Velasco G, LopezOtin C (1996) Molecular cloning of a novel membrane-type matrix metalloproteinase from a human breast carcinoma. Cancer Res 56:944-949[Abstract]
Pyke C, Ralfkiaer E, Tryggvason K, Danø K (1993) Messenger RNA for two type IV collagenases is located in stromal cells in human colon cancer. Am J Pathol 142:359-365[Abstract]
Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370:61-65[Medline]
StetlerStevenson WG, Liotta LA, Kleiner DE, Jr (1993) Role of matrix metalloproteinases in tumor invasion and metastasi. FASEB J 7:1434-1441
Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI (1995) Mechanism of cell surface activation of 72-kDa type IV collagenase. J Biol Chem 270:5331-5338
Takino T, Sato H, Shinagawa A, Seiki M (1995) Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. J Biol Chem 270:23013-23020
Terranova VP, Hujanen ES, Martin GR (1986) Basement membrane and the invasive activity of metastatic tumor cells. J Natl Cancer Inst 77:311-316[Medline]
Will H, Hinzmann B (1995) cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment. Eur J Biochem 231:602-608[Abstract]
Zucker S, Lysik RM, Wieman J, Wilkie DP, Lane B (1985) Diversity of human pancreatic cancer cell proteinases: role of cell membrane metalloproteinases in collagenolysis and cytolysis. Cancer Res 45:6168-6178[Abstract]
Zucker S, Moll UM, Lysik RM, DiMassimo EI, StetlerStevenson WG, Liotta LA, Schwedes JW (1990) Extraction of type-IV collagenase/gelatinase from plasma membranes of human cancer cells. Int J Cancer 45:1137-1142[Medline]