(Pump-1), Secreted from Human Rectal Carcinoma Cell Line1

[CANCER RESEARCH 50, 7758-7764, December 15. 1990] Purification and Characterization of Extracellular Matrix-degrading Metalloproteinase, Matrin (Pump-1), Secreted from Human Rectal Carcinoma Cell Line1

Kaoru Miyazaki,2 Yasuhisa Hattori, Fuminori Umenishi, Hidetaro Yasumitsu, and Makoto Umeda

Division of Cell Biology, Kifiara Institute for Biological Research, Yokohama City University, 2-120-3 Nakamura-cho, Minami-ku, Yokohama, Kanagawa 232, Japan

ABSTRACT nases (14-18), serine proteinases (19-22), thiol proteinases (23-25), and aspartic proteinases (26). Among the four classes A metalloproteinase with M, 29,000 was purified to homogeneity as a of proteinases, the metalloproteinases appear to play a major latent proenzyme from the conditioned medium of a human rectal carci role in matrix degradation. Recent studies have revealed a noma cell line CaR-1. This enzyme hydrolyzed casein more potently than family of secreted zinc metalloproteinases capable of degrading gelatin embedded in polyacrylamide gels in zymography assay. Calcium ECM proteins. These include interstitial collagenase (27-29), ion was essential for the activity. It exerted the maximum activity at pH 7-9. Its activity was stimulated by organomercurials, such as p-amino- type IV collagenases (or gelatinases) with M, 72,000 (16, 17) phenyl mercuric acetate and p-chloromercuric benzoic acid, and was and M, 92,000 (18), and stromelysin (transin) (30-34). The inhibited by 1,10-phenanthroline but was hardly affected by diisopropyl interstitial collagenase preferentially hydrolyzes the interstitial fluorophosphate and pepstatin. When the purified proenzyme was acti collagens of types I, II, and III, whereas type IV collagenases vated by the organomercurials, it effectively hydrolyzed fibronectin, do hydrolyze type IV basement membrane collagen but not type laminin, type IV basement membrane collagen, and several types of I collagen (16-18, 27). Stromelysin has a more broad substrate gelatins but not interstitial type I and III collagens. The treatment of the specificity: it hydrolyzes fibronectin, laminin, type IV collagen, purified proenzyme with /7-aminophenyl mercuric acetate or trypsin and proteoglycans (30). These metalloproteinases are structur formed an active peptide with M, 20,000. The structural analysis indi cated that it was most likely identical to putative metalloproteinase-1, ally related to each other and are secreted as latent proenzymes (zymogens) from various types of cells including tumor cells the complementary DNA of which had been cloned from human tumor mRNAs capable of hybridizing to a rat transin complementary DNA. and normal connective tissue cells. In addition to these en Based on the fact that this enzyme is secreted extracellularly and degrades zymes, Woessner and Talpin (35) recently isolated a metallo the matrix proteins, we propose the name "matrin" for this newly proteinase with M, 28,000 from postpartum rat uterus, desig identified enzyme. nated MMP-7, which was capable of degrading casein, fibro nectin, and various types of gelatins. On the other hand, Muller et al. (36) cloned cDNAs of two kinds of putative metallopro INTRODUCTION teinases, stromelysin-2 and pump-1, from human tumors with ECM,3 which consists of fibrous structural proteins, glyco- the use of transin cDNA as a probe. Pump-1 has an exception ally small molecular size (267 amino acid residues) compared proteins, proteoglycans, and glycosaminoglycans, plays a fun to other mammalian metalloproteinases. Recently, Quantin et damental role in the formation and maintenance of tissue al. (37) expressed the pump-1 cDNA in COS cells and dem architecture. The major structural proteins, collagens, support onstrated that the recombinant pump-1 protein was a precursor the basic structure of ECM, while the major adhesive glycopro- form of a metalloproteinase with a substrate specificity similar teins, fibronectin and laminin, mediate the binding of cells to to that of MMP-7. They also showed that pump-1-related ECM (1-3). These ECM proteins affect proliferation, differ mRNA was expressed in postpartum rat uteri, suggesting that entiation, morphology, substrate attachment, and motility of pump-1 might correspond to rat MMP-7. However, the native cells in vitro (2, 4). There is increasing evidence indicating that form of pump-1 has not been identified in human sources. proteolytic degradation of ECM is required for tumor cells to invade basement membranes, stremai matrix, and cell-cell junc We previously found that transformation of a rat liver epi tions (5-7). Liotta et al. (8, 9) and other groups (10, 11) have thelial cell line, BRL, with Rous sarcoma virus induced marked secretion of a fibronectin-degrading metalloproteinase (38). demonstrated that the secretion of type IV collagen-degrading Recently, we surveyed culture media conditioned by more than enzymes by tumor cells is well correlated with the invasive 30 kinds of nonmalignant and malignant cell lines for secreted potentials. Furthermore, in vitro invasion experiments have suggested that a cascade of serine proteinases and metallopro- proteinases by the zymography technique. As a result, it has been found that a human rectal adenocarcinoma cell line se teinases is required for tumor invasion into the basement mem cretes a low molecular weight metalloproteinase capable of branes (12, 13). degrading ECM proteins. In the present paper, we report the It has been reported that tumor cells secrete metalloprotei- purification of this metalloproteinase, tentatively named "ma trin," and its characterization. The structural analysis of the Received 2/19/90; accepted 8/30/90. The costs of publication of this article were defrayed in part by the payment purified enzyme demonstrated that it might be identical to the of page charges. This article must therefore be hereby marked advertisement in putative metalloproteinase pump-1. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. 2To whom requests for reprints should be addressed. MATERIALS AND METHODS 3The abbreviations used are: ECM, extracellular matrix; APMA, p-aminophenyl mercuric acetate; CBB, Coomassie brilliant blue R-250; DFP, diisopropyl Cells and Culture. A human rectal carcinoma cell line CaR-1 fluorophosphate; HPLC, high performance liquid chromatography; PAGE, poly (JCRB0207), which had been established from a metastatic lymph node acrylamide gel electrophoresis; PCMB, /7-chloromercuric benzoic acid; SDS, of a 70-year-old male patient with primary rectal adenocarcinoma by sodium dodecyl sulfate; MMP-7, matrix metalloproteinase-7; pump-1, putative metalloproteinase-1; cDNA, complementary DNA; DME, Dulbecco's modified Kaneko et al. (39), was used as the source of the low molecular weight Eagle's medium; FI2, Ham's F-12 medium. metalloproteinase matrin. Other human cancer cell lines tested for the 7758

secretion of the metalloproteinase were HLE (hepatoma), A549 (lung Zymography of Proteinases. Caseinolytic activities of secreted pro- adenocarcinoma), T24 (bladder carcinoma), EJ-1 (bladder carcinoma), teinases were analyzed by zymography according to the methods of A431 (vulva epidermoid carcinoma), HeLa.S3 (cervix epitheloid carci Chin et al. (30) with some modifications. SDS-polyacrylamide gels noma), HSC-4 (tongue squamous carcinoma), T98G (glioblastoma), containing 1 mg/ml casein were prepared with 1.3 mg/ml ammonium YKG-1 (glioma), YST-2 (schwannoma), YST-3 (schwannoma), persulfate (2.7-fold the usual amount). Samples to be tested were mixed HT1080 (fibrosarcoma), NB-1 (neuroblastoma), VMRC-MELG (mel with an equal volume of the concentrated SDS sample buffer [4% (w/ anoma), and RPMI8226 (myeloma). These human cell lines, except v) SDS, 125 HIM Tris-HCl (pH 6.8), 10% (v/v) glycerol] and then A549, YKG-1, YST-2, YST-3, and NB-1, and an epithelial cell line electrophoresed on the casein-containing gels without heating the mix derived from an African green monkey kidney BSC-1 were provided by ture in boiling water. After electrophoresis, the proteinases separated the Japanese Cancer Research Resource Bank. on the gels were renatured by gently shaking the gels in 2.5% Triton These cells were cultured at 37Â°Cina humidified atmosphere of 5% X-100 containing 50 mM Tris-HCl (pH 7.5) and 0.1 M NaCl at room CO2 and 95% air. The basal medium (DME/F12) consisted of a 1:1 temperature for l h to remove SDS, followed by incubation in 250 ml mixture of Dulbecco's modified Eagle's medium (GIBCO, Grand Is of 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl2 and 0.02% NaN3 land, NY) and Ham's F-12 medium (GIBCO), which was supplemented at 37Â°Cfor about 18 h. The resultant gels were stained with CBB and with 15 mM AL2-hydroxyethylpiperazine-A''-ethanesulfonic acid, 1.2 destained as described above. mg/ml of NaHCOj, 100 units/ml of penicillin G, and 0.1 mg/ml of Preparative SDS-PAGE. For structural analysis, the metalloprotei streptomycin sulfate. Cells were maintained in DME/F12 supple nase obtained from the Cellulofine GCL2000 column was further mented with 10% PCS (GIBCO) (10% PCS + DME/F12). Plastic purified by preparative SDS-PAGE on 10% polyacrylamide slab gels culture wares were obtained from Becton, Dickinson Labware (Oxnard, (160 mm long, 150 mm wide, 1 mm thick). After the electrophoresis, CA). the gels were briefly stained with CBB, destained, and soaked in water Preparation of Conditioned Medium. CaR-1 cells were grown to for 30 min. The CBB-stained band of the metalloproteinase was excised confluence in roller bottles (175 cm2) containing 200 ml of 10% PCS from the gel and cut into about 5 x 5-mm pieces. The gel pieces were + DME/F12. The cultures were then rinsed twice with Hanks' balanced placed in a dialysis bag with a small volume of 10 HIMTris-HCl (pH salt solution and incubated in serum-free DME/F12 overnight. The 8) containing 0.1% SDS and dialyzed overnight against the same buffer medium was discarded and replaced with fresh serum-free DME/F12, at room temperature. The protein eluted from the gel pieces was and the cultures were continued. The serum-free conditioned medium precipitated by adding 4 volumes of cold acetone and leaving the mixture at -20Â°Covernight, collected by centrifugation, dissolved in a was harvested twice/week and clarified by sequential centrifugation at 800 x g for 15 min and at 15,000 x g for 30 min. The protein present small volume of 0.1% SDS, and subjected to reverse-phase HPLC on in the clarified conditioned medium was precipitated by 80% saturation a phenyl-5PW RP column (Tosoh Co.). The eluted protein was used of ammonium sulfate and collected by centrifugation at 15,000 x g for for analysis of NH2-terminal amino acid sequence and amino acid 30 min. The protein precipitate was dissolved in a small volume of 10 composition. mM Tris-HCl (pH 7.5) containing 0.5 M NaCl and 0.01% Brij 35, Structural Analysis. The NH2-terminal amino acid sequence of the dialyzed against the same buffer, and used as concentrated conditioned purified metalloproteinase (about 20 Â¿ig)wasanalyzed with an Applied medium. Biosystems gas-phase protein sequencer provided courtesy of Drs. R. Column Chromatographies. Molecular sieve chromatography was Muramatsu and S. Abe (Bioscience Research Laboratories, Nippon carried out on a Cellulofine GCL2000 column (2.6 x 98 cm) (Chisso Mining Co., Ltd., Saitama, Japan). The amino acid composition of the Co., Ltd., Tokyo, Japan), previously equilibrated with 10 mM Tris-HCl purified proteinase (25 Â¿tg)was analyzed with a Hitachi amino acid analyzer model 385 after hydrolysis in 6 N HC1 at 110Â°Cfor 22 h in (pH 7.5) containing 0.5 M NaCl and 0.01% Brij 35. The concentrated conditioned medium of CaR-1 cells was applied to the column and Toray Research Center, Inc. (Kamakura, Japan). eluted with the same buffer at a flow rate of about 25 ml/h. Determination of Protein Concentrations. Protein concentrations were Anion-exchange high performance liquid chromatography (HPLC) determined by the dye method with a Bio-Rad protein assay kit, using was carried out on a Shodex QA-824 column (8 x 75 mm; Showa bovine serum albumin as the standard. Denko Co., Ltd., Tokyo, Japan), preequilibrated with 10 mM Tris-HCl Reagents. Native type IV collagen purified from an EHS tumor was buffer (pH 7.5) containing 0.01% Brij 35. The proteinase pool from a kind gift from Dr. Y. Ohba (Bioscience Research Laboratories, the molecular sieve chromatography was dialyzed against the same Nippon Mining Co., Ltd.). Bovine plasma fibronectin, mouse laminin, buffer and applied to the column at a flow rate of 0.5 ml/min. The and bovine lens type IV collagen were purchased from Nitta Gelatin charged column was washed with the buffer and eluted with a linear Co., Ltd. (Tokyo, Japan), bovine skin type I collagen and bovine gradient of NaCl from 0 to 0.5 M in 40 ml of the Tris-HCl buffer at a placenta type III collagen from Koken Co., Ltd. (Tokyo, Japan), DFP flow rate of 1 ml/min. and PCM B from Wako Chemicals, Ltd. (Osaka, Japan), APMA from Molecular sieve HPLC was carried out on a TSK gel G3000SW Tokyo Kasei Co., Ltd. (Tokyo, Japan), and pepstatin from Peptide column (7.5 x 300 mm) (Tosoh Co., Ltd., Tokyo, Japan), preequili Institute, Inc. (Osaka, Japan), respectively. brated with 0.1 M sodium phosphate buffer (pH 7.0) containing 0.2 M NaCl and 0.01% Brij 35. The proteinase-containing fractions from the RESULTS anion-exchange HPLC were mixed, concentrated by ultrafiltration through a Diaflo YM-10 membrane (Amicon; W. R. Grace & Co., Purification of Matrin. When secreted proteinases were sur Danvers, MA), and then applied to the column at a flow rate of 0.5 veyed with various cultured cell lines by zymography on casein- ml/min. containing SDS-polyacrylamide gels, the conditioned medium SDS-PAGE. Unless otherwise noted, SDS-PAGE was carried out of a human rectal adenocarcinoma cell line CaR-1 showed a on 12.5% polyacrylamide slab gels (90 mm long, 90 mm wide, 0.75 caseinolytic band with M, 29,000 (Fig. 1). When a gelatin- mm thick) under nonreducing conditions according to the method of containing gel was used, the conditioned medium showed a Laemmli (40) with a Bio-Rad electrophoresis apparatus (Richmond, faint gelatinolytic band with M, 29,000, while showing strong CA). The molecular weight markers used are rabbit muscle phospho- bands with M, 66,000-90,000. This indicated that casein was a rylase b (M, 97,400), bovine serum albumin (M, 66,200), hen egg albumin (M, 42,700), bovine carbonic anhydrase (M, 29,000), soybean better substrate than gelatin for the M, 29,000 proteinase. The trypsin inhibitor (M, 20,100), and hen egg lysozyme (M, 14,300). After strong gelatinolytic bands seemed due to the gelatinases char electrophoresis, the gels were stained in 0.25% (w/v) CBB containing acterized by other groups (16-18). The caseinolytic activity 45% (v/v) ethanol and 10% (v/v) acetic acid for 30 min. The stained with A/r 29,000 was hardly detected with the other 15 kinds of gels were destained briefly with a mixture of 25% ethanol and 8% acetic human cancer cell lines tested. However, a nonmalignant epi acid and then with a mixture of 5% ethanol and 7.5% acetic acid. thelial cell line from an African green monkey kidney, BSC-1, 7759

97k- Structural Analysis. In order to clarify the relationship of matrin with other known proteinases, its NH2-terminal amino acid sequence was analyzed (Fig. 6). The sample for analysis 66k- was obtained by using preparative SDS-PAGE after the first molecular sieve chromatography. This alternative purification 43k- procedure gave pure matrin with a high yield (0.3 mg/liter) but as a denatured form. The NH2-terminal amino acid sequence to the 25th amino acid residue was almost perfectly consistent with an NH2-terminal moiety (residues 18-42) of the putative metalloproteinase pump-1 (35). This strongly suggested that 29k- matrin might be identical to pump-1. The amino acid compo sition of the purified matrin was also in good agreement with that of a pump-1 peptide (residues 18-267) without the 17 NH2-terminal amino acids, supporting the above possibility (Table 1). Properties of Matrin. The optimum pH of matrin was ex 1 2 3 amined by incubating the casein-containing gels at various pHs Fig. 1. Analysis of proteinases secreted from CaR-1 and BSC-1 cells by after electrophoresis and subsequent renaturation of the puri zymography on casein- and gelatin-containing gels. The conditioned media from the serum-free cultures of CaR-1 (lanes I and 2) and BSC-1 (lane 3) were fied matrin. It exerted the maximum activity in a pH range of concentrated 30-fold by ammonium sulfate precipitation and dialyzed against 10 7-9. mM Tris-HCl (pH 7.5) containing 0.01% Brij 35. A 10-^1aliquot of each dialyzed Effects of various proteinase inhibitors on the activity of material was applied to a polyacrylamide gel containing 1 mg/ml casein (lanes 1 and 'I or gelatin (lane 2) after SDS treatment. Other experimental conditions matrin were examined using the zymography assay (Fig. 7). are described in the text. Ordinate, molecular weight in thousands. The activity of matrin was significantly increased by PCMB (a thiol proteinase inhibitor) but hardly affected by DFP (a serine secreted a high activity with a similar molecular weight. The proteinase inhibitor) and pepstatin (an aspartic proteinase in M, 29,000 proteinase in the CaR-1-conditioned medium also hibitor). One mM 1,10-phenanthroline completely inhibited the showed a proteolytic band on a fibronectin-containing gel (data proteinase activity even in the presence of 10 mM Ca2+, sug not shown), suggesting that it may be a matrix-degrading en gesting that this enzyme had a metal ion other than Ca2+ at the zyme. We tentatively named this enzyme matrin, and its puri active site. Matrin showed no activity in a reaction mixture fication was attempted from the serum-free conditioned me without Ca2+. These results indicate that matrin belongs to the dium of CaR-1 cells. metalloproteinase family. CaR-1 cells were cultured in a serum-free DME/F12 me In general, mammalian metalloproteinases are secreted as dium. Eight liters of the conditioned medium was concentrated latent proenzymes (zymogens) and unable to hydrolyze sub by ammonium sulfate precipitation and subjected to molecular strates unless activated by organomercurial compounds, pro sieve chromatography on a Cellulofine GCL2000 column (Fig. teinases, or some other reagents (17,18, 28, 30). In zymography 2A). The resultant fractions were analyzed by SDS-PAGE and assays, however, these proenzymes are able to hydrolyze sub proteinase zymography (Fig. 2B). The caseinolytic activity with strates embedded in polyacrylamide gels without the above- M, 29,000 was mostly eluted in fractions 57-65. The analysis mentioned treatments. This seems to be caused by the confor- by SDS-PAGE showed a CBB-stained protein band with M, mational changes of the proenzymes induced by SDS-PAGE 29,000 in these fractions, which corresponded to the caseinol and subsequent renaturation procedure. In the case of matrin, ytic activity in the zymogram. it hydrolyzed casein embedded in polyacrylamide gels in the The active fractions (fractions 59-63) from the Cellulofine zymography assay, but it could not hydrolyze casein in a free GCL2000 column were combined, dialyzed against 10 mw Tris- reaction mixture containing 50 mM Tris-HCl (pH 7.5) and 10 HCl buffer (pH 7.5) containing 0.01% Brij 35 and then sub mM CaCl2 unless an organomercurial compound, APMA or jected to anion-exchange HPLC on a Shodex QA-824 column PCMB, was supplemented to the mixture. This indicated that (Fig. 3). About 70% of the M, 29,000 proteinase was adsorbed matrin was also secreted as a latent proenzyme. When the to the column and eluted at approximately 0.1 M NaCl before purified proenzyme was treated with 1 HIM APMA in the presence of 10 mM CaCl2 at 30Â°Cfor l h and then analyzed by other adsorbed proteins, while the remainder passed through the column without adsorption. SDS-PAGE, it was converted to a peptide with MT 20,000 and The proteinase fractions (fractions 7-10) eluted from the another trace peptide with M, 23,000 (Fig. 8). Such lower Shodex QA-824 column were combined, concentrated by ultra- molecular weight forms of caseinolytic activities were also filtration, and finally subjected to molecular sieve HPLC on a observed in the zymograms of crude matrin preparations during TSK gel G3000SW column (Fig. 4). The proteinase activity purification. Therefore, the autolytic peptide with Mr 20,000 was eluted as a major peak with an apparent molecular weight seemed to be an active form of matrin, while the M, 23,000 of lower than 10,000. The abnormally retarded elution effec peptide seemed to be its intermediate form. Similar products tively separated this proteinase from contaminating proteins. were also obtained when the proenzyme was treated with 1 mM Thus, the secreted proteinase matrin was purified to homoge PCMB or with a small amount of trypsin. neity by the simple three-step procedure (Fig. 5). About 0.7 mg Substrate specificity of matrin against five kinds of ECM of matrin was obtained from 8 liters of the CaR-1 cell-condi proteins was examined in the reaction mixture supplemented tioned medium with a 110-fold enrichment. The electrophoretic with 1 mM APMA (Fig. 9). Matrin effectively hydrolyzed mobility of the purified enzyme was not affected by the presence fibronectin and laminin. A relatively short incubation with of 2-mercaptoethanol, indicating that it is a single-chain pep- matrin converted fibronectin and laminin to partially digested tide. fragments, but prolonged incubation caused their complete 7760

4 - o 00 CM

Fig. 2. Molecular sieve chromatography of concentrated conditioned medium of CaR-1 cells on Cellulofine GCL2000 column. Eight 2 - liters of the serum-free conditioned medium was concentrated to 30 ml and divided into two equal portions. Each portion, which con tained 280 mg of protein, was applied to the column. A. elution pattern of proteins in the chromatography; O, A2M value of each frac tion; arrows, elution positions of ferritin (M, 450,000), albumin (M, 66,000), and cytochrome c (M, 12,500); bar, fractions 59-63 from the two runs of the chromatography were 20 40 60 pooled and used for further purification. Other experimental conditions are given in the text. Fraction number (6 ml each) B, SDS-PAGE (left) and proteinase zymography on casein-containing gel (right) of the frac B (Proteinases) tions obtained from the molecular sieve col (Proteins) umn. Fr., fraction; ordinate, molecular weight in thousands; arrows, proteinase band with M, 29,000 in fractions 57-65. 97k- 66k- 43k-

29k-

20k-

Fr. 41 45 49 53 57 61 65 41 45 49 53 57 61 65

Fig. 3. Anion-exchange HPLC on Shodex QA-824 column of matrin obtained by molecu lar sieve chromatography. The proteinase pool from the Cellulofine GCL2000 column (Fig. 2), which contained 20 mg of protein, was dialyzed against 10 ITIMTris-HCl (pH 7.5) containing 0.01% Brij 35 and applied to the column as described in the text. Inset, SDS-PAGE profiles of the column fractions; abscissa, fractions 6- 15; ordinate, molecular weight in thousands. 6 8 10 14 The proteinase-containing fractions 7-10 were mixed and used for further purification.

0.0 10 20 Fraction number (0.5 ml each) 7761

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. METALLOPROTEINASE SECRETED BY HUMAN RECTAL CARCINOMA digestion. Among two subunits of laminili, the A chain was Table 1 Amino acid compositions of matrin and pump-1 more susceptible than the B chain. Matrin significantly de Matrin Pump-lÂ° graded native and pepsin-treated type IV basement membrane Amino acid mol% No." mol% No. collagens but hardly affected interstitial collagens of type I and Asp +AsnThrSerGlu type III. Although matrin showed a very low proteolytic activity on gelatin-containing gels (Fig. 1), it digested types I, III, and IV gelatins dissolved in the reaction mixtures (data not shown). +GinGlyAlaValCysMetIleLeuTyrPheLysHisArgProTrp(Total)11.254.776.429.2510.286.774.200.273.024.339.304.504.586.273.114.225.791.67(100)28121623261711181123111116811IS4(252)10.84.87.28.410.06.44.40.43.24.48.84.44.46.83.24.46.02.0(100)27121821251611181122111117811155(250)

0.5 290k 142k 67k 32k 12.4k

0.4

0.2

0.1 Â°The amino acid composition of the possible proenzyme form (residues 18- 0.0 267) of pump-1 (Ref. 36). 10 15 * Estimated number of amino acids/matrin molecule. Elution volume (ml) Fig. 4. Molecular sieve HPLC on TSK gel G3000SW column of matrin obtained by anion-exchange HPLC. The proteinase pool obtained from the Shodex QA-824 column (Fig. 3) was concentrated to 1 ml by ultrafiltration and divided into two equal portions. Each portion, which contained approximately 2 mg protein, was applied to the TSK gel G3000SW column as described in the text. Top abscissa, elution positions of molecular weight markers (in thousands): glutamate dehydrogenase (290,000), lÃ¡clate dehydrogenase (142,000), enolase (67,000), adenylate kinase (32,000), and cytochrome c (12,400); bar, protein peak was collected and used as purified matrin.

29k-

123456 Fig. 7. Effects of proteinase inhibitors on caseinolytic activity of purified matrin. The purified proteinase was electrophoresed on a casein-containing gel, and the resultant gel was renatured and cut into strips, followed by incubation in the Ca2*-containing (lanes 1-5) or Ca:*-free (lane 6) reaction mixture with or without the indicated proteinase inhibitor. Lane I, no addition; lane 2, 5 mM DFP; lane 3, 1 mM PCMB; lane 4, 5 Mg/ml pepstatin; lane 5, 1 mM 1,10- phenanthroline; lane 6, no addition (â€”Ca2*). Ordinate, molecular weight in thousands. Other conditions are the same as given in the text. The apparent doublet of the caseinolytic activity is an artifact caused by the CBB staining of the enzyme protein at the center of the protein band.

1 2 3 DISCUSSION Fig. 5. SDS-PAGE of matrin preparation from each purification step. Lane 1, concentrated conditioned medium; lane 2, from first molecular sieve chroma- In the present study, we isolated and characterized a new tography; lane 3, from anion-exchange HPLC; lane 4, from molecular sieve member of the matrix-degrading metalloproteinase family from HPLC (pure matrin). Ordinate, molecular weight in thousands. the conditioned medium of a human rectal adenocarcinoma cell line, CaR-1. This proteinase was secreted as a latent proenzyme 1 23456789 10 Leu Pro Leu Pro Gin Glu Ala Gly Gly Met with A/r 29,000 and converted to an active form with M, 20,000 by treatment with organomercurials or trypsin. The activated 11 12 13 14 15 16 17 18 19 20 enzyme required Ca2+ for the activity and appeared to have Ser Glu Leu Gin Trp (Glu) Gin Ala X (Asp) another metal ion at the active site. Like stromelysin, the 21 22 23 24 25 purified enzyme degraded various ECM proteins. It effectively Tyr Leu Lys Arg Phe hydrolyzed fibronectin, laminili, type IV basement membrane Fig. 6. NH2-terminal amino acid sequence of electrophoretically purified collagen, and several types of gelatins but hardly type I and III matrin. Parentheses, possible but not conclusive amino acids. The unidentified collagens. 19th amino acid corresponds to the 36th amino acid Gin in pump-1. Other amino acids including those in parentheses are consistent with the sequence of an NH2- The NH2-terminal amino acid sequence and amino acid terminal moiety (residues 18-42) in pump-1. composition of the purified M, 29,000 proenzyme strongly 7762

molecular weight value of the rat uterus enzyme estimated by SDS-PAGE was 35,000 under nonreducing conditions and 28,000 under reducing conditions, whereas the electrophoretic mobility of the CaR-1-derived enzyme (M, 29,000) was not affected by the presence or absence of a reducing reagent. Further studies are required to explain these discrepancies and to establish their identity. -29k So far the following four members of the matrix-degrading metalloproteinase family have been characterized by both pri mary structures and enzymological properties: interstitial collagenase, type IV collagenases (gelatinases) with M, 72,000 and -20k 92,000, and stromelysin. The CaR-1-derived enzyme is the fifth member of the metalloproteinase family. With the exception of interstitial collagenase, these metalloproteinases can more or less hydrolyze type IV collagen and other ECM proteins (17, 18, 30, 31). Therefore, the nomenclature of these proteinases seems rather contradictory or confusing. More systematic no Fig. 8. Effect of APMA treatment on molecular size of matrin. The purified menclature should be considered when their enzymological proteinase was incubated with (+) and without (â€”)1HIMAPMA at 30Â°Cfor 60 properties and functions are well established. The name "pump- min and then analyzed by SDS-PAGE. Ordinate, molecular weight in thousands. 1" seems unsuitable for the CaR-1-derived enzyme, since it is derived from a putative metalloproteinase. At present, we pro suggest that it is identical to pump-1 reported by Muller et al. pose to refer to the CaR-1 cell-derived metalloproteinase as (36). Like other mammalian metalloproteinases such as colla- matrin. genases and stromelysin, the cDNA-derived primary structure In general, the matrix-degrading metalloproteinases are of pump-1 contains a putative zinc-binding site homologous to highly produced by connective tissue cells. For example, normal the zinc-binding sequence of thermolysin. Therefore, it is very human skin fibroblasts are known to constitutively secrete likely that the purified enzyme is a zinc metalloproteinase. interstitial collagenase and stromelysin in vitro (31 ). Although Pump-1 was recently suggested to correspond to the rat uterus it seems doubtless that these enzymes play an essential role in metalloproteinase (MMP-7), which had been purified from the the maintenance and remodeling of ECM, their roles in tumor extract of postpartum rat uteri by Woessner and Talpin (35). growth and invasion remain to be clarified. Matrisian et al. (32, These three enzymes share common properties such as molec 33) found that transcription of a gene encoding an unknown ular weight values of the precursor and active forms, optimum protein, termed transin, was induced when fibroblasts were pH and Ca2"1"requirement for activity, but differences also exist transformed by polyomavirus, Rous sarcoma virus, or H-ras among them. MMP-7 and recombinant pump-1 were unable to oncogene or treated with epidermal growth factor. Later, tran digest type IV collagen (36, 37), whereas the CaR-1-derived sin was shown to be a rat homologue of human stromelysin enzyme effectively digested the basement membrane collagen. (34). We have reported that a rat liver epithelial cell line, BRL, It seems possible that this difference was derived from the secretes a high activity of a fibronectin-degrading metallopro difference in the experimental conditions such as the amounts teinase when transformed with Rous sarcoma virus (38). Re and specific activities of the used enzymes. In addition, the cently, this proteinase was purified and identified as transin

12 34 5678 9 10 11 12 Fig. 9. Substrate specificity of purified ma trin. Substrate proteins were incubated with (+) or without (â€”)10 Mg/ml of the purified matrin in SOjil of a reaction mixture of 20 HIM Tris-HCl (pH 7.5), 10 mM CaCU, 0.01% Brij 200k- 35, and 1 mM APMA at 37Â°Cfor 6 h (lanes 1-4) or at 30Â°Cfor 18 h (lanes 5-12). After the incubation, the reaction mixtures were mixed with the SDS sample buffer containing 116k- 5% 2-mercaptoethanol, heated in boiling water for 3 min, and subjected to SDS-PAGE on 97k- 7.5% polyacrylamide gels. Lanes 1 and 2, 0.2 mg/ml bovine plasma fibronectin; lanes 3 and 4, 0.2 mg/ml mouse laminin; lanes 5 and 6, 0.5 mg/ml pepsin-treated bovine skin type I collagen; lanes 7 and 8, 0.5 mg/ml pepsin- 66k- treated bovine placenta type III collagen; lanes 9 and 10, 0.5 mg/ml pepsin-treated bovine lens type IV collagen; lanes 11 and 12, 0.5 mg/ml EHS tumor-derived type IV collagen. Arrow heads, partially digested fragments of fibronec tin (M, (ordinate) 220,000, 150,000, 88,000, 43k- 68,000, 50,000, 38,000, 36,000, and 32,000] and laminin (M, 170,000, 150,000, 58,000, and 34,000). - + - 7763

(41). These results suggest the involvement of this metallopro- 17. Collier, I. E., Wilhelm, S. M., Eisen, A. Z., Marmer, B. L., Grant, G. A., Seltzer, J. L., Kronberger, A., He, C., Bauer, E. A., and Goldberg, G. I. H- teinase in the malignant transformation of some types of cells. ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete Muller et al. (36) have surveyed 50 kinds of human primary a single metalloprotease capable of degrading basement membrane collagen. tumors for the expression of stromelysin-related genes and J. Biol. Chem., 263: 6579-6587, 1988. 18. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., detected the mRNA of stromelysin or stromelysin-2 in 6 tu and Goldberg, G. I. SV40-transformed human lung fibroblasts secrete a 92- mors, all of which are squamous carcinomas. Recently, we kDa type IV collagenase which is identical to that secreted by normal human macrophages. J. Biol. Chem., 264: 17213-17221, 1989. examined proteinase species secreted from various cultured cell 19. Unkeless, J., DaÃ±o,K., Kellerman, G. M., and Reich, E. Fibrinolysis asso lines using proteinase zymography on casein- and gelatin-con ciated with oncogenic transformation. Partial purification and characteriza tion of the cell factor, a plasminogen activator. J. Biol. Chem., 249: 4295- taining gels. Among 16 kinds of human cancer cell lines tested, 4303, 1974. type IV collagi-naso with M, 72,000 was significantly secreted 20. Wilson, E. L., Becker, M. L. B., Hoal, E. G., and Dowdle, E. B. Molecular from 13 cell lines, type IV collagi-nasi- with M, 92,000 from 6 species of plasminogen activators secreted by normal and neoplastic human cell lines, stromelysin-like metalloproteinase from 4 cell lines, cells. Cancer Res., 40: 933-938, 1980. 21. Gaylis, F. D., Keer, H. N., Wilson, M. J., Kwaan, H. C., Sinha, A. A., and and matrin from only one cell line.4 These results suggest that Kozlowski, J. M. Plasminogen activators in human prostate cancer cell lines type IV collagi-nasi- with M, 72,000 is the most common and tumors: correlation with the aggressive phenotype. J. Urol., 142: 193- 198, 1989. metalloproteinase in human tumors, whereas matrin is re 22. Kao, R. T., Wong, M., and Stern, R. Elastin degradation by proteinases from stricted to specific cell types. Matrin-like metalloproteinase is cultured human breast cancer cells, Biochem. Biophy. Res. Commun., 105: 383-389, 1982. also secreted from the African green monkey kidney cell line, 23. Poole, A. R., Tiltman, K. J., Recklies, A. D., and Stocker, T. A. M. BSC-1. Therefore, matrin may play a more specific role than Differences in secretion of the proteinase cathepsin B at the edge of human other metalloproteinases, although the possibility cannot be breast carcinomas and fibrosarcomas. Nature (Lond.) 273: 545-549, 1980. 24. Sloane, B. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. Purification and Characterization of Extracellular Matrix-degrading Metalloproteinase, Matrin (Pump-1), Secreted from Human Rectal Carcinoma Cell Line

Kaoru Miyazaki, Yasuhisa Hattori, Fuminori Umenishi, et al.

Cancer Res 1990;50:7758-7764.

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