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Activation of Human Meprin-α in a Cell Culture Model of Colorectal Cancer Is Triggered by the Plasminogen-activating System

2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês

10.1074/jbc.m206203200

1083-351X

Sandra Rösmann, Dagmar Hahn, Daniel Lottaz, Markus-N. Kruse, Walter Stöcker, Erwin E. Sterchi,

Blood Coagulation and Thrombosis Mechanisms

The activation of latent proenzymes is an important mechanism for the regulation of localized proteolytic activity. Human meprin-α, an astacin-like zinc metalloprotease expressed in normal colon epithelial cells, is secreted as a zymogen into the intestinal lumen. Here, meprin is activated after propeptide cleavage by trypsin. In contrast, colorectal cancer cells secrete meprin-α in a non-polarized way, leading to accumulation and increased activity of meprin-α in the tumor stroma. We have analyzed the activation mechanism of promeprin-α in colorectal cancer using a co-culture model of the intestinal mucosa composed of colorectal adenocarcinoma cells (Caco-2) cultivated on filter supports and intestinal fibroblasts grown in the companion dish. We provide evidence that meprin-α is activated by plasmin and show that the presence of plasminogen in the basolateral compartment of the co-cultures is sufficient for promeprin-α activation. Analysis of the plasminogen-activating system in the co-cultures revealed that plasminogen activators produced and secreted by fibroblasts converted plasminogen to active plasmin, which in turn generated active meprin-α. This activation mechanism offers an explanation for the observed meprin-α activity in the tumor stroma, a prerequisite for a potential role of this protease in colorectal cancer. The activation of latent proenzymes is an important mechanism for the regulation of localized proteolytic activity. Human meprin-α, an astacin-like zinc metalloprotease expressed in normal colon epithelial cells, is secreted as a zymogen into the intestinal lumen. Here, meprin is activated after propeptide cleavage by trypsin. In contrast, colorectal cancer cells secrete meprin-α in a non-polarized way, leading to accumulation and increased activity of meprin-α in the tumor stroma. We have analyzed the activation mechanism of promeprin-α in colorectal cancer using a co-culture model of the intestinal mucosa composed of colorectal adenocarcinoma cells (Caco-2) cultivated on filter supports and intestinal fibroblasts grown in the companion dish. We provide evidence that meprin-α is activated by plasmin and show that the presence of plasminogen in the basolateral compartment of the co-cultures is sufficient for promeprin-α activation. Analysis of the plasminogen-activating system in the co-cultures revealed that plasminogen activators produced and secreted by fibroblasts converted plasminogen to active plasmin, which in turn generated active meprin-α. This activation mechanism offers an explanation for the observed meprin-α activity in the tumor stroma, a prerequisite for a potential role of this protease in colorectal cancer. Human meprin is a metalloprotease of the astacin family of proteases (1Dumermuth E. Sterchi E.E. Jiang W.P. Wolz R.L. Bond J.S. Flannery A.V. Beynon R.J. J. Biol. Chem. 1991; 266: 21381-21385Google Scholar, 2Stöcker W. Grams F. Baumann U. Reinemer P. Gomis-Rüth F.X. McKay D.B. Bode W. Protein Sci. 1995; 4: 823-840Google Scholar, 3Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Google Scholar). Meprins were first isolated from brush-border membranes of mammalian intestinal and kidney epithelial cells (4Sterchi E.E. Green J.R. Lentze M.J. Clin. Sci. (Lond.). 1982; 62: 557-560Google Scholar, 5Sterchi E.E. Green J.R. Lentze M.J. J. Pediatr. Gastroenterol. Nutr. 1983; 2: 539-547Google Scholar, 6Beynon R.J. Shannon J.D. Bond J.S. Biochem. J. 1981; 199: 591-598Google Scholar, 7Kenny A.J. Ingram J. Biochem. J. 1987; 245: 515-524Google Scholar). The protease is a multidomain glycoprotein consisting of evolutionarily related α- and β-subunits with subunit masses of 85–110 kDa (8Sterchi E.E. Naim H.Y. Lentze M.J. Hauri H.P. Fransen J.A. Arch. Biochem. Biophys. 1988; 265: 105-118Google Scholar,9Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Google Scholar). The subunits form disulfide-linked dimers, which can further oligomerize by noncovalent interactions, leading to homo- or hetero-oligomeric proteins (8Sterchi E.E. Naim H.Y. Lentze M.J. Hauri H.P. Fransen J.A. Arch. Biochem. Biophys. 1988; 265: 105-118Google Scholar, 9Marchand P. Tang J. Bond J.S. J. Biol. Chem. 1994; 269: 15388-15393Google Scholar, 10Ishmael F.T. Norcum M.T. Benkovic S.J. Bond J.S. J. Biol. Chem. 2001; 276: 23207-23211Google Scholar). Meprins are synthesized as type I transmembrane proteins; however, only the β-subunit has been determined to be an integral membrane protein (11Johnson G.D. Hersh L.B. J. Biol. Chem. 1994; 269: 7682-7688Google Scholar). The α-subunit is processed post-transcriptionally after the C-terminally located I-domain, resulting in detachment of its membrane-spanning anchor (12Marchand P. Tang J. Johnson G.D. Bond J.S. J. Biol. Chem. 1995; 270: 5449-5456Google Scholar). Thus, when expressed individually, meprin-α is secreted from the cells; when both subunits are expressed, meprin-α associates with meprin-β at the cell surface (11Johnson G.D. Hersh L.B. J. Biol. Chem. 1994; 269: 7682-7688Google Scholar). Despite their similar protein domain structures, meprin-α and meprin-β show a clear difference in substrate specificity (13Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Google Scholar). According to a variety of known in vitro substrates, meprins may be implicated in normal and pathologic processes. They degrade proteins of the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; u-PA, urokinase-type plasminogen activator; t-PA, tissue-type plasminogen activator; u-PAR, urokinase-type plasminogen activator receptor; PAI, plasminogen activator inhibitor; PET, polyethylene terephthalate; MDCK, Madin-Darby canine kidney; PABA peptide, N-benzoyl-l-tyrosyl-p-aminobenzoic acid. 1The abbreviations used are: ECM, extracellular matrix; u-PA, urokinase-type plasminogen activator; t-PA, tissue-type plasminogen activator; u-PAR, urokinase-type plasminogen activator receptor; PAI, plasminogen activator inhibitor; PET, polyethylene terephthalate; MDCK, Madin-Darby canine kidney; PABA peptide, N-benzoyl-l-tyrosyl-p-aminobenzoic acid. such as type IV collagen, fibronectin, laminin, and nidogen (14Kaushal G.P. Walker P.D. Shah S.V. J. Cell Biol. 1994; 126: 1319-1327Google Scholar, 15Walker P.D. Kaushal G.P. Shah S.V. Kidney Int. 1998; 53: 1673-1680Google Scholar) and process biologically active peptides, including bradykinin, angiotensins, parathyroid hormone, gastrin, the β-chain of insulin, growth factors, and cytokines (8Sterchi E.E. Naim H.Y. Lentze M.J. Hauri H.P. Fransen J.A. Arch. Biochem. Biophys. 1988; 265: 105-118Google Scholar, 13Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Google Scholar, 16Chestukhin A. Litovchick L. Muradov K. Batkin M. Shaltiel S. J. Biol. Chem. 1997; 272: 3153-3160Google Scholar). Like other astacins, human meprin is initially synthesized as an inactive precursor protein that is activated after proteolytic processing of the N-terminal prosequence (17Bode W. Gomis-Rüth F.X. Huber R. Zwilling R. Stöcker W. Nature. 1992; 358: 164-167Google Scholar). In vitro, trypsin cleaves the propeptide of human meprin following an Arg residue (Arg65 in human meprin-α and Arg61 in human meprin-β) (18Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Google Scholar), and this is most probably the physiologic mechanism of promeprin activation in the intestinal lumen. However, possible other meprin-activating proteases must exist in vivo, especially in meprin-expressing tissues or cells, where trypsin is absent. Because meprin has also been detected in leukocytes of the subepithelial intestinal mucosa and in a variety of cancer cells and tissues (19Matters G.L. Bond J.S. Mol. Carcinog. 1999; 25: 169-178Google Scholar, 20Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133Google Scholar), knowledge of these proteases is an important factor to determine the physiologic significance of meprin activity under normal and pathologic conditions. In this study, we demonstrate that plasmin activates promeprin-α. Plasmin is a trypsin-like protease present in the blood and the extracellular fluid as inactive plasminogen. Conversion of plasminogen to active plasmin is catalyzed by the urokinase-type plasminogen activator (u-PA) or tissue-type plasminogen activator (t-PA). Although t-PA is generally accepted to be primarily involved in thrombolysis, it is mainly u-PA that generates active plasmin in processes leading to ECM breakdown (21Andreasen P.A. Kjøller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Google Scholar). Several proteases, including serine proteases and metalloproteases, are implicated in tumor growth, invasion into surrounding tissues, and metastasis. Plasmin, through the direct degradation of proteins of the basement membrane and the extracellular matrix (22Mackay A.R. Corbitt R.H. Hartzler J.L. Thorgeirsson U.P. Cancer Res. 1990; 50: 5997-6001Google Scholar, 23Goldfinger L.E. Stack M.S. Jones J.C. J. Cell Biol. 1998; 141: 255-265Google Scholar, 24Vassalli J.D. Sappino A.P. Belin D. J. Clin. Invest. 1991; 88: 1067-1072Google Scholar) or the activation of zymogen forms of ECM-degrading metalloproteases such as interstitial procollagenase (matrix metalloproteinase-1) and prostromelysin (matrix metalloproteinase-3) (25He C.S. Wilhelm S.M. Pentland A.P. Marmer B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Google Scholar), plays a crucial part in these tissue-remodeling processes. Cell-surface activation of plasminogen is tightly controlled and is mediated through the concerted action of u-PA, the plasminogen activator receptor (u-PAR), and plasminogen activator inhibitors (PAI-1 and PAI-2) (26Andreasen P.A. Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Google Scholar). In the small intestine, both meprin-α and meprin-β are localized in the brush-border membranes of epithelial cells. In the colon, only meprin-α is expressed and is secreted apically into the colon lumen (27Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Google Scholar). We have previously shown that, in contrast to normal colonocytes, colorectal adenocarcinoma cells (Caco-2) secrete meprin-α in a non-polarized way to the apical and basolateral media when grown on Transwell filter dishes (20Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133Google Scholar). Moreover, we have observed an increased activity of meprin-α in colorectal cancer tissue homogenates and a diffuse cellular localization in colon cancer cells by immunohistochemistry (20Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133Google Scholar). Other authors have described an up-regulation of the expression of u-PA in colorectal carcinomas and its correlation with tumor invasion (28Yang J.L. Seetoo D. Wang Y. Ranson M. Berney C.R. Ham J.M. Russell P.J. Crowe P.J. Int. J. Cancer. 2000; 89: 431-439Google Scholar, 29Hansen T.P. Fenger C. Kronborg O. Acta Pathol. Microbiol. Immunol. Scand. 1999; 107: 689-694Google Scholar). Using immunohistochemistry and in situ hybridization, u-PA and u-PAR expression was shown to be localized to colon epithelial cells (30Harvey S.R. Sait S.N., Xu, Y. Bailey J.L. Penetrante R.M. Markus G. Am. J. Pathol. 1999; 155: 1115-1120Google Scholar), stromal fibroblasts, and endothelial and/or inflammatory cells (for a review, see Ref. 21Andreasen P.A. Kjøller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Google Scholar). We therefore speculated that the plasminogen-activating system plays an important role in meprin-α activation in colorectal cancer tissue, in the subepithelial mucosa, or in other meprin-expressing cells and tissues in vivo. To test this hypothesis, we established a co-culture model of the intestinal mucosa by cultivating Caco-2 cells on Transwell filter inserts together with primary intestinal fibroblasts or HT-1080 fibrosarcoma cells in the companion dish. We analyzed meprin-α protein expression and secretion in differentiated Caco-2 cells and demonstrate that addition of the proenzyme plasminogen to the basolateral compartment of the co-cultures was sufficient for meprin-α activation. We examined components of the plasminogen-activating system in the co-cultures and propose a model for meprin-α activation in the subepithelial mucosa and colon cancer stroma in vivo. Cell culture media and all supplements were obtained from Invitrogen (Basel, Switzerland). Enzyme inhibitors, bovine fibrinogen, trypsin, and thrombin were purchased from Sigma. Human plasmin, bovine and human plasminogen, pure high molecular mass u-PA, one-chain t-PA, monoclonal antibody against u-PA (no. 3689), and substrates used for plasmin and plasminogen activator assays were from American Diagnostica. All chemicals for gel electrophoresis were obtained from Bio-Rad. Polyclonal antibodies recognizing promeprin-α and both promeprin-α and its mature form were raised in rabbits by immunization with a glutathione S-transferase fusion protein from a 421-amino acid N-terminal sequence (31Dumermuth E. Eldering J.A. Grünberg J. Jiang W. Sterchi E.E. FEBS Lett. 1993; 335: 367-375Google Scholar) and a 118-amino acid fragment of the intervening domain (27Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Eur. J. Biochem. 1999; 259: 496-504Google Scholar). Caco-2 cells at passages 40–50 were grown in HEPES-buffered minimal essential medium supplemented with 20% (v/v) fetal calf serum, 4.5 g/liter d-glucose, nonessential amino acids (100 μm each), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine at 37 °C in 95% air and 5% CO2. Cells were seeded at a density of 6 × 105 on culture plate inserts, comprising a membrane with a 4.2-cm2 growth area (polyethylene terephthalate (PET) membranes, 0.4-μm pore size, BD Biosciences; or polycarbonate membranes, Millicell, Millipore AG). Inserts were placed in six-well plates and incubated in 2.5 ml of apical medium and 3 ml of basolateral medium. The media were changed daily. Establishment of tight monolayers was followed by light microscopy and measurement of the transepithelial electrical resistance at room temperature (Millicell-ERS, Millipore AG). A transepithelial electrical resistance of >250 ohms × cm2 indicated confluence. Primary intestinal fibroblasts were derived from an intestinal mucosal biopsy that was cultured in HEPES-buffered minimal essential medium, 10% (v/v) fetal calf serum, 200 μg/ml chlorotetracycline, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mmglutamine. For experiments, intestinal fibroblasts at passages 4–10 were used. HT-1080 fibrosarcoma cells were grown in the same medium as described for intestinal fibroblasts with addition of 1× nonessential amino acids (100 μm each) and omission of chlorotetracycline. Madin-Darby canine kidney (MDCK) cells stably transfected with human meprin-α cDNA (32Eldering J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Google Scholar) were grown in HEPES-buffered minimal essential medium supplemented with 5% (v/v) fetal calf serum, 400 μg/ml Geneticin, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. Confluent cells were incubated for 24 h in serum-free medium. The conditioned medium was removed, sterile-filtered, and stored at −20 °C until further usage. 5 × 105 primary intestinal fibroblasts or HT-1080 cells were seeded on six-well companion plates and grown for 24 h until confluence. Caco-2 cells were grown separately for 7 days post-confluence on filter supports as described above. Cells were washed twice with serum-free medium to remove fetal calf serum. Inserts with Caco-2 cells were then assembled with the fibroblasts grown on the companion plates and incubated for 20 h in 2 ml of serum-free medium in each compartment. Human plasminogen was added to the basolateral medium as indicated. The culture media from the apical and basolateral compartments were collected, sterile-filtered, and stored at −20 °C until further usage. Cells were washed twice with phosphate-buffered saline, scraped with a rubber spatula, and harvested by centrifugation. The sedimented cells were suspended in phosphate-buffered saline containing protease inhibitors (2 μg/ml pepstatin, 2 μg/ml aprotinin, 5 μg/ml leupeptin, 3.5 μg/ml benzamidine, and 1.7 mmphenylmethylsulfonyl fluoride) and lysed in 1% (v/v) Nonidet P-40 and 1% (w/v) sodium deoxycholate for 30 min on ice. Cellular debris was removed by centrifugation, and the supernatant is referred to as the cell lysate. Protein samples were subjected to electrophoresis under reducing or nonreducing conditions on 7.5 or 10% SDS-polyacrylamide gels (33Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) and transferred to a polyvinylidene difluoride membrane (Hybond P, Amersham Biosciences). The membrane was saturated with 5% dry milk and 0.1% Tween 20 in Tris-buffered saline for 2 h, incubated overnight with the indicated antibody (polyclonal anti-meprin-α antibody, 1:2000–1:5000 dilution; or monoclonal anti-u-PA antibody, 1:1000 dilution), and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:10,000 dilution) for 1.5 h at room temperature. Immunocomplexes were visualized using the ECL Plus Western blotting kit (Amersham Biosciences) and x-ray films. The activity of plasminogen activators was determined by fibrin zymography as described previously (34Choi N.S. Kim S.H. Anal. Biochem. 2000; 281: 236-238Google Scholar). Bovine fibrin (1.2 mg/ml) and plasminogen (15 μg/ml) were copolymerized on a 10% SDS-polyacrylamide gel. 15-μl aliquots of conditioned medium were subjected to electrophoresis under nonreducing conditions. The gel was washed with 2.5% Triton X-100 for 1 h at room temperature to remove SDS, followed by 16 h of development in 30 mmTris-HCl, 200 mm NaCl, and 0.02% NaN3 (pH 7.4) at 37 °C. The gel was stained and fixed with 0.1% Coomassie Blue R-250 in methanol/acetic acid/water (40:10:50). Total RNA was isolated from cultured cells using the acid guanidinium/thiocyanate method according to the manufacturer's recommendations (Trizol reagent, Invitrogen). First-strand cDNA synthesis was carried out with 1 μg of RNA and random hexanucleotide primers using the GeneAmp® RNA PCR kit (Applied Biosystems). The amount of cDNA in each sample was quantified using PCR with primers for the glyceraldehyde-3-phosphate dehydrogenase gene as an internal standard. The oligonucleotide primers employed for the PCRs were as follows: meprin-α(5′), 5′-GGGACATCCTCTTGCAGAAATCC-3′; meprin-α(3′), 5′-CAACGAGTCT GTCTGAAGGACTTC-3′; u-PA(5′), 5′-GGAGGGCAGCACTGTGAAATAG-3′; u-PA(3′), 5′-AAGGCAATGTCGTTGTGGTGAG-3′; t-PA(5′), 5′-TGGAGCAGTCTTCGTTTCGC-3′; t-PA(3′), 5′-CCCATTCCCAAAGTAGCAGTCAC-3′; u-PAR(5′, 5′-TGTAAGACCAACGGGGATTGC-3′; u-PAR(3′), 5′-CGGTGGCTACCAGACATTGATTC-3′; PAI-1(5′), 5′-TTTGGTGAAGGGTCTGCTGTGC-3′; PAI-1(3′), 5′-CGTCTGATTTGTGGAAGAGGCG-3′; PAI-2(5′), 5′-CAGAAGGGTAGTTATCCTGATGCG-3′; PAI-2(3′), 5′-GCTGGTCCACTTGTTGAGTTTGTC-3′; GAPDH(5′), 5′-ACATCAAGAAGGTGGTGAAGCAGG-3′; and GAPDH(3′), 5′-CTCTTCCTCTTGTGCTCTTGCTGG-3′. PCR conditions were as follows: 2 min at 95 °C; 35 cycles for 0.5 min at 95 °C, 0.5 min at 56–58 °C, and 1.5 min at 72 °C; and 10 min at 72 °C. Prior to assaying, cells were suspended in ice-cold phosphate-buffered saline containing 0.1% Triton X-100 and lysed by sonification for 1 min at 4 °C. Alkaline phosphatase activity was measured at room temperature by following the hydrolysis of p-nitrophenyl phosphate with a commercially available kit (Roche Molecular Biochemicals, Basel). Sucrase-isomaltase activity was assayed in cell extracts using the method of Dahlqvist (35Dahlqvist A. Scand. J. Clin. Lab. Invest. 1984; 44: 169-172Google Scholar). Meprin-α activity was measured in the culture media after zymogen activation with trypsin or plasmin usingN-benzoyl-l-tyrosyl-p-aminobenzoic acid (PABA peptide) as a substrate and analyzed as described previously (8Sterchi E.E. Naim H.Y. Lentze M.J. Hauri H.P. Fransen J.A. Arch. Biochem. Biophys. 1988; 265: 105-118Google Scholar). u-PA activity was determined by measuring the increase in absorbance of the free chromophore p-nitroanilide generated after hydrolysis of carbobenzoxy-l-Glu(α-t-butyl-ester)-Gly-Arg-p-nitroanilide (SPECTROZYME® UK, no. 244, American Diagnostica). For the assay, cells were grown for 20 h in serum-free medium (free of colored matter). Enzyme activity was measured in sterile-filtered medium according to the manufacturer's instructions using high molecular mass u-PA as an activity standard. Purification of plasmin-activated meprin-α was achieved according to the procedure described for purification of trypsin-activated meprin-α (36Köhler D. Kruse M.-N. Stöcker W. Sterchi E.E. FEBS Lett. 2000; 465: 2-7Google Scholar) with a slight modification. Recombinant promeprin-α (500 μg), purified by gel filtration from overexpressing High Five insect cell culture medium, was incubated with 10 nm human plasmin in 0.1 m Tris-HCl, 0.1m NaCl, 0.1% polyethylene glycol 8000 (pH 7.5) for 5 h at room temperature. The activated form of meprin-α was loaded onto an affinity column carrying the immobilized inhibitor Pro-Leu-Gly-hydroxamate, which selectively binds active meprin-α. After elution, protein fractions with the highest meprin-α activity were pooled, precipitated, and subjected to SDS-PAGE. Following transfer to a polyvinylidene difluoride membrane, the Coomassie Blue-stained meprin-α band was excised and subjected to N-terminal amino acid sequencing (SeqLab). We have previously shown a non-polarized secretion of meprin-α in Caco-2 cells (20Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133Google Scholar). To analyze this further, Caco-2 cells were grown on PET membrane inserts over a period of 21 days post-confluence. During this period, Caco-2 cells differentiate spontaneously to a polarized phenotype (37Mariadason J.M. Rickard K.L. Barkla D.H. Augenlicht L.H. Gibson P.R. J. Cell. Physiol. 2000; 183: 347-354Google Scholar). Establishment of tight monolayers was judged by light microscopy and measurement of the transepithelial electrical resistance. At 7 days post-confluence, the transepithelial electrical resistance reached a level of 700–750 ohms × cm2, a value that is generally accepted as proof of a tight cell layer. Apical and basolateral media as well as cell lysates at different days post-confluence were analyzed for meprin-α protein expression by immunoblotting (Fig. 1). A plateau of maximal meprin-α expression was reached at 7 days post-confluence (Fig. 1 A, lane 3). The higher molecular mass form of 100 kDa in cell extracts represents the endoplasmic reticulum resident precursor of meprin-α (18Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Google Scholar), whereas the 90-kDa protein corresponds to the C-terminally processed form of meprin-α (38Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Google Scholar). We confirmed that the 95-kDa secreted form of meprin-α was directed in a non-polarized way to the apical and basolateral compartments (Fig.1 B). In accordance with expression levels, a maximal level of total meprin-α secretion was reached at 7 days post-confluence (Fig. 1 B, lanes 5 and 6). More than 50% of the enzyme reached the basolateral compartment at this time, as verified by densitometric scanning analysis. Although secretion of meprin-α into the apical medium remained constant during the residual culture period, the amount of basolaterally secreted meprin-α diminished in the later phases of cell culture (Fig. 1 B,lanes 10 and 12). This is most likely the result of hindrance of basolateral transport due to the formation of cell multilayers, a phenomenon that has been described for prolonged cultivation of Caco-2 cells on PET filters (39Rothen-Rutishauser B. Braun A. Günthert M. Wunderli-Allenspach H. Pharm. Res. (N. Y.). 2000; 17: 460-465Google Scholar). When Caco-2 cells were grown on polycarbonate membranes (Millicell), ∼50% of meprin-α was secreted basolaterally throughout the 21-day culture period (Fig.1 C, lanes 8, 10, and 12). Thus, cells cultivated for >10 days post-confluence on PET filters were prone to "filter clogging." To analyze the phenotypic development of cell polarization during the culture period, the activity of two marker enzymes of the brush-border membrane, alkaline phosphatase and sucrase-isomaltase, were assayed in the Caco-2 cell extracts. Alkaline phosphatase and sucrase-isomaltase activities both increased progressively, reaching a 3-fold increase at 7 days post-confluence, further demonstrating that cells had reached a differentiated state (data not shown). In conclusion, meprin-α protein expression is induced in differentiated Caco-2 cells, and targeting and secretion of the enzyme are clearly independent of cell polarization. Taking all these results into account, plus the fact that cells may be monitored by light microscopy on PET filters, we used 7-day post-confluent Caco-2 cells grown on these filter inserts for the subsequent co-cultures experiments. Following zymogen activation by limited trypsin treatment, meprin-α hydrolytic activity was measured using the PABA peptide as a substrate (8Sterchi E.E. Naim H.Y. Lentze M.J. Hauri H.P. Fransen J.A. Arch. Biochem. Biophys. 1988; 265: 105-118Google Scholar). To investigate whether plasmin activates promeprin-α, the medium of MDCK cells stably transfected with meprin-α cDNA (32Eldering J.A. Grünberg J. Hahn D. Croes H.J. Fransen J.A. Sterchi E.E. Eur. J. Biochem. 1997; 247: 920-932Google Scholar) was incubated with various concentrations of human plasmin or trypsin ranging from 10 to 500 nm for 2 h at 37 °C. Measurement of PABA peptide-hydrolyzing activity revealed that active meprin-α was generated in a dose-dependent manner after plasmin and trypsin treatment (Fig. 2). Plasmin showed no hydrolytic activity for the PABA peptide itself (not shown). The activities of trypsin-treated meprin-α were higher than those after plasmin treatment, indicating that activation by trypsin was more efficient. Processing of promeprin-α by plasmin was also assessed by immunoblotting. The zymogen was incubated with pure human plasmin or trypsin (50 nm) for 0–6 h at 37 °C. Immunodetection using an anti-meprin-α antibody directed against both the zymogen and mature enzyme (Fig. 2 B, upper panel) revealed a protein band corresponding to the zymogen (95 kDa) and an additional slightly smaller band after 3 and 6 h of incubation with plasmin. Detection with an antibody recognizing only the zymogen (Fig.2 B, lower panel) showed that this form disappeared with incubation time, confirming that promeprin-α was converted to mature meprin-α by plasmin treatment. Activation of meprin-α with trypsin (50 nm) for 3 h led to a quantitative conversion of promeprin-α to the mature form. To map the cleavage site, plasmin-activated meprin-α was subjected to N-terminal sequence analysis. Recombinant meprin-α (500 μg) isolated from the culture medium of meprin-α-overexpressing High Five insect cells was incubated with human plasmin (10 nm) for 5 h at room temperature. The activated form of meprin-α was then purified by affinity chromatography, followed by SDS-PAGE and transfer to a polyvinylidene difluoride membrane as described under "Experimental Procedures." The N-terminal sequence obtained corresponds to the sequence previously reported for trypsin-activated meprin-α (18Grünberg J. Dumermuth E. Eldering J.A. Sterchi E.E. FEBS Lett. 1993; 335: 376-379Google Scholar). Hence, plasmin and trypsin cleave promeprin-α at the same site, between Arg65 and Asn66, thereby generating mature enzymes with identical N termini. Non-polarized secretion of meprin-α from colon cancer cells, increased meprin-α activity measured in colon cancer tissue homogenates (20Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Cancer Res. 1999; 59: 1127-1133Google Scholar), and up-regulation of components involved in plasminogen activation described in colon adenocarcinomas (28Yang J.L. Seetoo D. Wang Y. Ranson M. Berney C.R. Ham J.M. Russell P.J. Crowe P.J. Int. J. Cancer. 2000; 89: 431-439Google Scholar) led us to speculate that the plasminogen-activating system may play a role in the activation of basolaterally secreted meprin-α in colon cancer tissue. Addressing this issue, we established an in vitroco-culture model of the intestinal mucosa composed of Caco-2 cells grown on filter supports and primary intestinal fibroblasts grown to confluence in the bottom companion dish (see model in Fig. 8). We investigated whether basolaterally secreted meprin-α was activated if plasminogen was added to the lower compartment of the co-cultures (Fig.3). In addition to primary intestinal fibroblasts that were isolated from an intestinal mucosal biopsy, HT-1080 fibrosarcoma cells were used in the co-culture experiments. The latter are known to express elevated levels of plasminogen activators (40Quax P.H. van Leeuwen R.T. Verspaget H.W. Verheijen J.H. Cancer Res. 1990; 50: 1488-1494Google Scholar). The presence of plasminogen in the basolateral medium of the co-cultures resulted in a dose-dependent activation of meprin-α in this compartment, whereas no activity was measured in the absence of plasminogen (Fig. 3 A). Addition of plasminogen to the apical compartment did not lead to activation of apically secreted meprin-α. This, plus the fact that addition of plasminogen to the basolateral medium of Caco-2 cells cultured alone did not lead to promeprin-α activation, clearly indic