Development of In Situ Zymography to Localize Active Matrix Metalloproteinase-7 (Matrilysin-1)
Advanced Core Technology Laboratories, Fuji Photo Film Co., Ltd., Kanagawa, Japan (RN,MY,HA) and Department of Pathology, School of Medicine, Keio University, Tokyo, Japan (FK,GH,TS,YO)
Correspondence to: Yasunori Okada, MD, PhD, Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan. E-mail: okada{at}sc.itc.keio.ac.jp
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Summary |
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Key Words: matrix metalloproteinase-7 Matrilysin-1 in situ zymography proteolytic activity tissue localization carcinoma invasion
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Introduction |
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We have previously developed gelatin-based film in situ zymography (FIZ-GN), which uses uniform thin layer of crosslinked gelatin on polyethylene terephthalate support films. FIZ-GN was applied to many tissue samples and detected gelatinolytic activity within tissues under various pathophysiological conditions, such as cancers of the brain (Nakada et al. 1999), thyroid (Nakamura et al. 1999
), oral cavity (Shimada et al. 2000
), esophagus (Koyama et al. 2000
), liver (Kaneyoshi et al. 2001
) and kidney (Kamiya et al. 2003
), and nonneoplastic diseases including rheumatoid arthritis (Yamanaka et al. 2000
), cardiovascular diseases (Nishikawa et al. 2003
), autoimmune renal disease (Alport syndrome) (Rao et al. 2003
), and ultraviolet damage of skin (Inomata et al. 2003
). This method was suitable to localize gelatinolytic activities (i.e., mainly MMP-2 and MMP-9, in the tissues) and used to analyze the effect of synthetic MMP inhibitors within cancer tissues (Ikeda et al. 2000
; Wada et al. 2003
; Yamamoto et al. 2003
). On the other hand, similar in situ zymography using the quenched fluorogenic substrate DQ-gelatin has been developed (Mook et al. 2003
). The method is considered to be superior in the sensitivity and quantification for the gelatinolytic activity of MMP-2 and MMP-9, because fluorescence is generated by cleavage of DQ-gelatin at sites of gelatinolytic activity (Frederiks and Mook 2004
). However, there are so far no such methods to localize MMP-7 activity within tissues.
In the present study, we attempted to develop a new in situ zymography for MMP-7 by preparing a suitable substrate of MMP-7. Because most of the MMP-7 substrates, including type IV collagen, gelatin, fibronectin, laminin, aggrecan (Imai et al. 1995), entactin (Sires et al. 1993
), and tenascin (Imai et al. 1994
), are also degraded by other MMPs, and some substrates are difficult to obtain in a large scale, we focused on carboxymethylated transferrin (Cm-Tf), which was originally developed as a substrate of MMP-3 (Okada et al. 1986
), but preferably digested by MMP-7 (Imai et al. 1995
). We have finally succeeded in developing film in situ zymography using crosslinked Cm-Tf (CCm-Tf) substrate to localize MMP-7 activity in human endometrial carcinoma and lung carcinoma tissues.
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Materials and Methods |
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Preparations of Proteinase Solutions
MMP-1, -2, -3, -7, -9, and -13; membrane-type 1 MMP (MT1-MMP = MMP-14); MT3-MMP (MMP-16); and ADAMTS4 (aggrecanase-1) were purified according to previous methods (Okada et al. 1986; Okada et al. 1990
; Okada et al. 1992
; Imai et al. 1995
; Knauper et al. 1996
; Ohuchi et al. 1997
; Shimada et al. 1999
; Nakamura et al. 2000
). These MMPs, except for MT1-MMP, MT3-MMP, and ADAMTS4, were activated by incubation with p-aminophenylmercuric acetate, and their concentrations were titrated using tissue inhibitors of metalloproteinase-1 or tissue inhibitors of metalloproteinase-2. Human liver cathepsin B, human liver cathepsin D, bovine kidney cathepsin H, and human liver cathepsin L (Calbiochem; San Diego, CA) were adjusted to 1.6 U/ml with 20 mM acetate buffer (pH 5.0) containing 1 mM EDTA and 200 mM NaCl. Bovine pancreas chymotrypsin (Biogenesis; Dorset, UK), human plasmin (American Diagnostica Inc.; Stamford, CT), porcine pancreas elastase (Sigma-Aldrich Japan; Tokyo, Japan), trypsin (Sigma-Aldrich Japan) and human neutrophil cathepsin G (Cortex Biochem; San Leandro, CA) were adjusted to 0.3, 0.5, 0.2, 8, and 2.7 U/ml with 50 mM Tris-HCl buffer (pH 7.5) containing 5 mM CaCl2, respectively.
Specificity Test of CCm-Tf Films to Proteinases
Specificity of CCm-Tf films against metalloproteinases was examined by incubation with 0.1 µM MMP-1, -2, -3, -7, -9, and -13; MT1-MMP; MT3-MMP; and ADAMTS4 in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 10 mM CaCl2 and 0.05% Brij-35. Two µl of each enzyme solution was spotted on the surface of CCm-Tf and FIZ-GN films. The films were incubated at 37C for 24 hr in a moist chamber, stained with staining solution containing 0.3% Biebrich Scarlet (Sigma-Aldrich Japan; Tokyo, Japan), 3.3% trichloroacetic acid and 50% ethanol in water for 6 min and then destained with water for 10 min. Specificity of CCm-Tf films against cathepsins and other serine proteinases was examined in a similar way.
Pretreatment of CCm-Tf Films with Proteinase Inhibitors
To inhibit serine proteinases (trypsin, chymotrypsin, and elastase), CCm-Tf films were pretreated with a solution of 100 µg/ml aprotinin (Wako Pure Chemical; Osaka, Japan) and 1.5 mg/ml elastatinal (Peptide Institute Inc.; Osaka, Japan) in water before use. For inhibition of MMPs or both MMPs and serine proteinases, the films were pretreated with 19.8 mg/ml 1,10-phenanthroline monohydrate (Sigma-Aldrich Japan), a mixture of 100 µg/ml aprotinin and 1.5 mg/ml elastatinal, or a mixture of 19.8 mg/ml 1,10-phenanthroline monohydrate, 100 µg/ml aprotinin, and 1.5 mg/ml elastatinal in 50% ethanol. A portion of the inhibitor solutions (125 µl) was put on a glass slide, and the coated side of a CCm-Tf film was applied to it. After incubation for 10 min, the CCm-Tf layer became fully swollen with the inhibitor solution. Then, the films were peeled off from the glass slide and dried spontaneously by holding them vertical. The pretreated films were stored in a refrigerator until use.
To evaluate the proteinase specificity of inhibitor-treated films, 2 µl of 0.1 µM active MMP-7, 0.2 U/ml porcine pancreas elastase, 8 U/ml bovine pancreas trypsin, and 2.7 U/ml cathepsin G in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 5 mM CaCl2 were spotted on the surface of CCm-Tf films, incubated at 37C for 20 hr and stained as described previously.
Reproducible Lysis of CCm-Tf Using Frozen Sections of MMP-7 Solution
Active MMP-7 was mixed with 20% aqueous polyvinylpyrrolidone solution at concentrations ranging from 3 to 400 µg/ml, snap-frozen in liquid nitrogen, and stored at 80C until used. The frozen sections (14 µm thick) were prepared with a cryostat microtome and mounted on CCm-Tf films. The films with sections were incubated at 37C for 24 hr in a moist chamber and then stained with staining solution for 4 min. An image of each film was taken by CCD camera and converted to monochrome using Adobe Photoshop (Adobe System Inc.; San Jose, CA). Then, the average optical density within the degradation spots was measured using ImageGauge (Fuji Photo Film; Tokyo, Japan).
In Situ Zymography for Frozen Sections of Human Carcinoma Tissues
Tissues of human endometrial adenocarcinoma (15 cases) and nonsmall-cell lung adenocarcinoma (five cases) were obtained from patients who underwent surgical resection at the Keio University Hospital, Tokyo, Japan. Written informed consent was obtained from each patient for the experimental use of the tissues. The samples were embedded without fixation in Tissue-Tek OCT compound (Sakura Finechemical Co. Ltd.; Tokyo, Japan), snap-frozen in liquid nitrogen, and stored at 80C until use. The serial frozen sections (6 µm thick) were prepared with a cryostat microtome and mounted on CCm-Tf films, which were prepared by coating with the aqueous Cm-Tf solution containing 1.6% crosslinking on polyethylene terephthalate support films and heat drying to crosslink the substrate layer as described previously. Immediately after mounting the sections on the films, the films were incubated at 37C for 816 hr in a moist chamber and then stained with staining solution containing 0.3% Biebrich Scarlet for 4 min. After washing for 10 min with water, the films were treated with Mayer's hematoxylin to counterstain nuclei for 2 min, washed for 10 min with water, and kept in 20% glycerol for 5 min. After drying the films spontaneously by keeping them vertical, they were cover-slipped using Tissue-Tek SCA cover slipping film (Sakura Finetek Japan; Tokyo, Japan) with a drop of xylene and imaged under optical microscope. The CCm-Tf substrate in contact with active MMP-7 within the tissue sections was digested, and thus zones of MMP-7 activity were negatively stained. Serial frozen sections were also subjected to in situ zymography using CCm-Tf films impregnated with 1,10-phenanthroline monohydrate, a mixture of aprotinin and elastatinal or a mixture of 1,10-phenanthroline monohydrate, aprotinin, and elastatinal.
Immunohistochemistry of MMP-7
Serial frozen sections were immunostained with monoclonal antibody against MMP-7 (141-7B2; Daiichi Fine Chemical Co. Ltd.; Takaoka, Japan) or nonimmune mouse IgG according to our previous method (Ueno et al. 1999). Briefly, the sections were mounted on 3-aminopropyl-triethoxysilane-coated slides and fixed with 4% paraformaldehyde. After blocking endogenous peroxidase and nonspecific binding with 0.3% H2O2 and 10% normal horse serum, the slides were reacted with 4 µg/ml antiMMP-7 antibody or 4 µg/ml nonimmune mouse IgG and then with an avidin-biotin-peroxidase complex (DAKO; Glostrup, Denmark). Color was developed with 0.03% 3,3'-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl buffer, pH 7.6 containing 0.006% H2O2. Counterstaining was performed with hematoxylin. Serial sections were also stained with hematoxylin and eosin.
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Results |
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Discussion |
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In contrast to CCm-Tf films, FIZ-GN using the crosslinked gelatin films was susceptible to the proteolysis by all the MMPs tested (MMP-1, -2, -3, -7, -9, and -13; MT1-MMP; and MT3-MMP), ADAMTS4, serine proteinases (trypsin, chymotrypsin, plasmin, elastase, and cathepsin G) and cysteine proteinases (cathepsin B, H, and L), but not by an aspartic proteinase (cathepsin D). These data indicate that gelatin is a nonspecific substrate of many proteinases, and crosslinking is not so effective in improving the selectivity. Thus FIZ-GN is useful for the detection of a variety of proteolytic activities in the tissues, but not suitable for the detection of specific MMP unless single MMP species is dominantly expressed in the tissue.
In situ zymography with quenched fluorogenic DQ-gelatin has been developed for detection of gelatinolytic activities of MMP-2 and MMP-9 (Mook et al. 2003). Because in this technique fluorescent peptides are produced by cleavage of DQ-gelatin at sites of gelatinolytic activity, the method appears to provide better tissue localization of the activity with higher sensitivity and quantification than FIZ-GN, in which staining intensity decreases at gelatinolytic areas (Frederiks and Mook 2004
). However, FIZ-GN and in situ zymography using CCm-Tf films have an advantage in that one can preserve the films after the incubation and take microscopic pictures of the digestion by an ordinary light microscope, whereas the fluorescent signals by in situ zymography using DQ-gelatin quickly disappear during observation by a confocal microscopy. McIntyre et al. (2004)
have recently developed a polymer-based fluorogenic substrate serving as a selective proteolytic beacon for MMP-7 activity and used the method for in vivo imaging by intravenous injection of the substrate. Although they mentioned in their article that the substrate could be used for in situ zymography for detection of MMP-7 activity in frozen sections of tumors (McIntyre et al 2004
), the method is not yet established. Thus, to our knowledge, our in situ zymography using CCm-Tf films is the only method to localize MMP-7 activity within tissues at the present time.
We have applied the newly developed in situ zymography for MMP-7 to localize its activity in the human endometrial and lung adenocarcinoma specimens. By using CCm-Tf films impregnated with inhibitors to serine proteinases (aprotinin and elastatinal) and metalloproteinases (1,10-phenanthroline), we could demonstrate the metalloproteinase activity in the carcinoma cell nests. Because the activity was colocalized with MMP-7 shown by immunohistochemistry, it is conceivable that the activity is derived from MMP-7 produced by the carcinoma cells. In general, living tissues contain many proteinases, some of which may degrade CCm-Tf. Therefore, combining the present method with other supplementary techniques (i.e., in situ zymography using inhibitor-containing CCm-Tf films and MMP-7 immunohistochemistry) is considered to be important to determine the MMP-7 activity within the tissues.
In summary, we have developed a new technique to detect MMP-7 activity that is applicable to localize the activity within the tissues. MMP-7 is upregulated during tumorigenesis (Takeuchi et al. 1997; Wilson et al. 1997
) and progression of carcinomas such as endometrial and gastrointestinal carcinomas (Yoshimoto et al. 1993
; Mori et al. 1995
; Ueno et al. 1999
). Thus we believe that the present technique is useful to study the role of MMP-7 in pathophysiological conditions such as tumorigenesis and cancer progression and to evaluate the effects of synthetic inhibitors to MMP-7 activity in vivo.
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Acknowledgments |
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Footnotes |
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