Activation of Pro-gelatinase B by Endometase/Matrilysin-2 Promotes Invasion of Human Prostate Cancer Cells
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m210975200
ISSN1083-351X
AutoresYun-Ge Zhao, Ai-Zhen Xiao, Robert G. Newcomer, Hyun I. Park, Tiebang Kang, Leland W.K. Chung, Mark G. Swanson, Haiyen E. Zhau, John Kurhanewicz, Qing-Xiang Amy Sang,
Tópico(s)Cell Adhesion Molecules Research
ResumoThis work has explored a putative biochemical mechanism by which endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) may promote human prostate cancer cell invasion. Here, we showed that the levels of MMP-26 protein in human prostate carcinomas from multiple patients were significantly higher than those in prostatitis, benign prostate hyperplasia, and normal prostate glandular tissues. The role of MMP-26 in prostate cancer progression is unknown. MMP-26 was capable of activating pro-MMP-9 by cleavage at the Ala93–Met94 site of the prepro-enzyme. This activation proceeded in a time- and dose-dependent manner, facilitating the efficient cleavage of fibronectin by MMP-9. The activated MMP-9 products generated by MMP-26 appeared more stable than those cleaved by MMP-7 under the conditions tested. To investigate the contribution of MMP-26 to cancer cell invasion via the activation of MMP-9, highly invasive and metastatic human prostate carcinoma cells, androgen-repressed prostate cancer (ARCaP) cells were selected as a working model. ARCaP cells express both MMP-26 and MMP-9. Specific anti-MMP-26 and anti-MMP-9 functional blocking antibodies both reduced the invasiveness of ARCaP cells across fibronectin or type IV collagen. Furthermore, the introduction of MMP-26antisense cDNA into ARCaP cells significantly reduced the MMP-26 protein level in these cells and strongly suppressed the invasiveness of ARCaP cells. Double immunofluorescence staining and confocal laser scanning microscopic images revealed that MMP-26 and MMP-9 were co-localized in parental and MMP-26 sense-transfected ARCaP cells. Moreover, MMP-26 and MMP-9 proteins were both expressed in the same human prostate carcinoma tissue samples examined. These results indicate that MMP-26 may be a physiological and pathological activator of pro-MMP-9. This work has explored a putative biochemical mechanism by which endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) may promote human prostate cancer cell invasion. Here, we showed that the levels of MMP-26 protein in human prostate carcinomas from multiple patients were significantly higher than those in prostatitis, benign prostate hyperplasia, and normal prostate glandular tissues. The role of MMP-26 in prostate cancer progression is unknown. MMP-26 was capable of activating pro-MMP-9 by cleavage at the Ala93–Met94 site of the prepro-enzyme. This activation proceeded in a time- and dose-dependent manner, facilitating the efficient cleavage of fibronectin by MMP-9. The activated MMP-9 products generated by MMP-26 appeared more stable than those cleaved by MMP-7 under the conditions tested. To investigate the contribution of MMP-26 to cancer cell invasion via the activation of MMP-9, highly invasive and metastatic human prostate carcinoma cells, androgen-repressed prostate cancer (ARCaP) cells were selected as a working model. ARCaP cells express both MMP-26 and MMP-9. Specific anti-MMP-26 and anti-MMP-9 functional blocking antibodies both reduced the invasiveness of ARCaP cells across fibronectin or type IV collagen. Furthermore, the introduction of MMP-26antisense cDNA into ARCaP cells significantly reduced the MMP-26 protein level in these cells and strongly suppressed the invasiveness of ARCaP cells. Double immunofluorescence staining and confocal laser scanning microscopic images revealed that MMP-26 and MMP-9 were co-localized in parental and MMP-26 sense-transfected ARCaP cells. Moreover, MMP-26 and MMP-9 proteins were both expressed in the same human prostate carcinoma tissue samples examined. These results indicate that MMP-26 may be a physiological and pathological activator of pro-MMP-9. extracellular matrix analysis of variance androgen repressed prostate cancer cells line benign prostate hyperplasia fibronectin integrated morphometry analysis matrix metalloproteinase-7/matrilysin matrix metalloproteinase-9/gelatinase B matrix metalloproteinase-26/endometase/matrilysin-2 matrix metalloproteinases 3-cyclohexylamino-1-propanesulfonic acid During the initial phases of carcinoma cell invasion, as tumor cells begin to spread and infiltrate into the surrounding normal tissues, these cells must first degrade the basement membrane and other elements of the extracellular matrix (ECM),1 including type IV collagen, laminin, and fibronectin (FN) (1Matrisian L.M. Bioessays. 1992; 14: 455-463Crossref PubMed Scopus (1329) Google Scholar). Multiple protease families, including the matrix metalloproteinases (MMPs), serine proteases, and cysteine proteases, are suspected of contributing to the invasive and metastatic abilities of a variety of malignant tumors (2Goldfarb R.H. Liotta L.A. Semin. Thromb. Hemostasis. 1986; 12: 294-307Crossref PubMed Scopus (137) Google Scholar, 3McCawley L.J. Matrisian L.M. Mol. Med. Today. 2000; 6: 149-156Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 4Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3226) Google Scholar, 5Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 163-176Crossref Scopus (5113) Google Scholar), but the specific biochemical mechanisms that facilitate these invasive behaviors remain elusive. More than 23 human MMPs, and numerous homologues from other species, have been reported (5Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 163-176Crossref Scopus (5113) Google Scholar), and matrix metalloproteinase-26 (MMP-26)/endometase/matrilysin-2 is a novel member of this enzyme family that was recently cloned and characterized by our group (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and others (7de Coignac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.P. Berthier O. Schmitt D. Bonnefoy J.Y. Gauchat J.F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (106) Google Scholar, 8Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar, 9Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 356: 705-718Crossref PubMed Scopus (135) Google Scholar). MMP-26 mRNA is primarily expressed in epithelial cancers, such as lung, breast, endometrial, and prostate carcinomas, in their corresponding cell lines (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 7de Coignac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.P. Berthier O. Schmitt D. Bonnefoy J.Y. Gauchat J.F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (106) Google Scholar, 8Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar, 9Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 356: 705-718Crossref PubMed Scopus (135) Google Scholar), and in a very limited number of normal adult tissues, such as the uterus (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 8Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar), placenta (7de Coignac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.P. Berthier O. Schmitt D. Bonnefoy J.Y. Gauchat J.F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (106) Google Scholar, 8Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar), and kidney (9Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 356: 705-718Crossref PubMed Scopus (135) Google Scholar). Recently, we have found that the levels of MMP-26 gene and protein expression are higher in a malignant choriocarcinoma cell line (JEG-3) than in normal human cytotrophoblast cells (10Zhang J. Cao Y.J. Zhao Y.-G. Sang Q.-X. Duan E.-K. Mol. Hum. Reprod. 2002; 8: 659-666Crossref PubMed Scopus (66) Google Scholar). Our preliminary studies indicate that expression of MMP-26 may be correlated with the malignant transformation of human prostate and breast epithelial cells. The specific expression of MMP-26 in malignant tumors and the proteolytic activity of this enzyme against multiple components of the ECM, including fibronectin, type IV collagen, vitronectin, gelatins, and fibrinogen, as well as non-ECM proteins such as insulin-like growth factor-binding protein 1 and α1-protease inhibitor (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 7de Coignac A.B. Elson G. Delneste Y. Magistrelli G. Jeannin P. Aubry J.P. Berthier O. Schmitt D. Bonnefoy J.Y. Gauchat J.F. Eur. J. Biochem. 2000; 267: 3323-3329Crossref PubMed Scopus (106) Google Scholar, 8Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar, 9Marchenko G.N. Ratnikov B.I. Rozanov D.V. Godzik A. Deryugina E.I. Strongin A.Y. Biochem. J. 2001; 356: 705-718Crossref PubMed Scopus (135) Google Scholar), indicate that MMP-26 may possess an important function in tumor progression. Another member of the MMP family considered to be an important contributor to the processes of invasion, metastasis, and angiogenesis exhibited by tumor cells is gelatinase B (MMP-9) (11Scorilas A. Karameris A. Arnogiannaki N. Ardavanis A. Bassilopoulos P. Trangas T. Talieri M. Br. J. Cancer. 2001; 84: 1488-1496Crossref PubMed Scopus (222) Google Scholar, 12Hrabec E. Strek M. Nowak D. Hrabec Z. Respir. Med. 2001; 95: 1-4Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 13Sakamoto Y. Mafune K. Mori M. Shiraishi T. Imamura H. Mori M. Takayama T. Makuuchi M. Int. J. Oncol. 2000; 17: 237-243PubMed Google Scholar, 14Shen K.H. Chi C.W. Lo S.S. Kao H.L. Lui W.Y. Wu C.W. Anticancer Res. 2000; 20: 1307-1310PubMed Google Scholar). Urı́a and López-Otı́n (8Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar) have demonstrated that MMP-26 is able to cleave MMP-9, and here we examine the possibility that MMP-26 facilitates tumor cell invasion through the activation of pro-MMP-9. The highly invasive and metastatic cell line utilized for this study, an androgen-repressed human prostate cancer (ARCaP), was derived from the ascites fluid of a patient with advanced prostate cancer that had metastasized to the lymph nodes, lungs, pancreas, liver, kidneys, and bones (15Zhau H.Y. Chang S.M. Chen B.Q. Wang Y. Zhang H. Kao C. Sang Q.A. Pathak S.J. Chung L.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15152-15157Crossref PubMed Scopus (191) Google Scholar). This cell line produces high levels of MMP-9 and gelatinase A (MMP-2) (15Zhau H.Y. Chang S.M. Chen B.Q. Wang Y. Zhang H. Kao C. Sang Q.A. Pathak S.J. Chung L.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15152-15157Crossref PubMed Scopus (191) Google Scholar, 16Matsubara S. Wada Y. Gardner T.A. Egawa M. Park M.S. Hsieh C.L. Zhau H.E. Kao C. Kamidono S. Gillenwater J.Y. Chung L.W.K. Cancer Res. 2001; 61: 6012-6019PubMed Google Scholar). In this study, we provide evidence that MMP-26 is capable of activating pro-MMP-9, and that once activated, MMP-9 cleaves fibronectin, type IV collagen, and gelatin with great efficiency. Both the MMP-26 and MMP-9 proteins were highly expressed in the ARCaP cells, and co-localization of their expression patterns was consistently observed. The invasiveness of ARCaP cells through FN or type IV collagen was significantly decreased in the presence of antibodies specifically targeting MMP-26 or MMP-9. In addition, cells transfected with antisense MMP-26, showing significant reduction of MMP-26 at the protein level, exhibited a reduction of invasive potential in vitro in addition to a significant diminution in observed levels of active MMP-9 protein. These results support the hypothesis that activation of MMP-9 by MMP-26 may promote the in vitroinvasiveness of ARCaP cells through FN or type IV collagen, whereas the co-expression of MMP-26 and MMP-9 in many human prostate carcinoma tissues indicates that this relationship may also occur in vivo. ARCaP, DU145, PC-3, and LNCaP, which are all established human prostate carcinoma cell lines, were routinely grown in low-glucose Dulbecco's modified Eagle′s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO2at 37 °C. Purified recombinant MMP-26 (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) or MMP-7 were incubated with purified pro-MMP-9 (17Sang Q.-X. Birkedal-Hansen H. Van Wart H.E. Biochim. Biophys. Acta. 1995; 1251: 99-108Crossref PubMed Scopus (95) Google Scholar) or pro-MMP-2 (18Sang Q.A. Bodden M.K. Windsor L.J. J. Protein Chem. 1996; 15: 243-253Crossref PubMed Scopus (41) Google Scholar) in HEPES buffer (50 mm HEPES, pH 7.5, 200 mm NaCl, 10 mm CaCl2, and 0.01% Brij-35) at 37 °C. For the dosage dependence of MMP-9 activation, MMP-9 (0.2 μm, final concentration) was incubated with MMP-7 and MMP-26 at the indicated molar concentration ratio (2:1, 4:1, and 8:1) for 24 h. The MMP-9 activation was quenched by 2× SDS-PAGE sample buffer containing 50 mm EDTA. The resulting solution was further diluted five times and 5 μl of the diluted sample was loaded onto SDS-polyacrylamide gels (8%). For the time dependence of MMP-9 activation, MMP-9 (0.2 μm) was incubated with MMP-7 (0.05 μm) and MMP-26 (0.05 μm) for the indicated time periods (0, 4, 8, 24 and 48 h) before quenching with the sample buffer. For FN cleavage assays, 2 μl of FN (0.25 mg/ml) were incubated with 30 μl of MMP-26 (final concentration 0.05 μm), pro-MMP-9 (final concentration 0.2 μm), or MMP-26-activated MMP-9 solutions in 1× HEPES buffer at 37 °C for 18 h. For silver staining, the reaction was stopped by adding 4× reducing sample buffer (6% SDS, 40% glycerol, 200 mm Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 200 mm EDTA, and 0.08% bromphenol blue) and boiled for 5 min. Following electrophoresis on a 9% SDS-polyacrylamide gel, the protein bands were visualized by silver staining (19Zhao Y.G. Wei P. Sang Q.-X. Biochem. Biophys. Res. Commun. 2001; 289: 288-294Crossref PubMed Scopus (15) Google Scholar). For gelatin zymogram, the gel was incubated for 3 h at 37 °C before it was stained with 0.1% Coomassie Blue solution (17Sang Q.-X. Birkedal-Hansen H. Van Wart H.E. Biochim. Biophys. Acta. 1995; 1251: 99-108Crossref PubMed Scopus (95) Google Scholar, 20Zhao Y.-G. Xiao A.Z. Cao X.M. Zhu C. Mol. Reprod. Dev. 2002; 62: 149-158Crossref PubMed Scopus (34) Google Scholar, 21Li H. Bauzon D.E. Xu X. Tschesche H. Cao J. Sang Q.-X. Mol. Carcinog. 1998; 22: 84-94Crossref PubMed Scopus (51) Google Scholar). Samples were separated by SDS-PAGE and transferred to ProBlottTM polyvinylidene difluoride membranes (Applied Biosystems) using CAPS buffer (10 mm CAPS, pH 11, 0.005% SDS). Proteins were visualized by staining with Coomassie Brilliant Blue R-250 solution (0.1% Coomassie Brilliant Blue R-250, 40% methanol, 1% acetic acid) and excised fragments were sent for sequencing. N-terminal sequencing was performed at the Bioanalytical Core Facility, Florida State University. RNA was extracted from the original cells by Trizol according to manufacturer protocols (Invitrogen, Carlsbad, CA), and 2 μg of total RNA were subjected to reverse transcriptase-PCR according to the standard protocol provided with the PCR kit (Invitrogen Corp., Carlsbad, CA). The MMP-26 forward primer was 5′-ACCATGCAGCTCGTCATCTTAAGAG-3′; the reverse primer was 5′-AGGTATGTCAGATGAACATTTTTCTCC-3′; for glyceraldehyde-3-phosphate dehydrogenase the forward primer was 5′-ACGGATTTGGTCGTATTGGG-3′; the reverse primer was 5′-TGATTTTGGAGGGATCTCGC-3′. PCR reactions were performed using a Biometra Personal Cycler (Biometra, Germany) with 30 thermal cycles of 10 s at 94 °C denaturing, 30 s at 60 °C annealing, and 1 min at 72 °C elongation. Ten μl of the amplified PCR products were then electrophoresed on a 1.0% agarose gel containing 0.5 mg/ml ethidium bromide for analysis of size differences. To confirm the amplification of the required cDNA sequences, PCR products were digested with a restriction enzyme as directed by the manufacturer. Specific antigen peptides corresponding to unique sequences in the pro-domain and metalloproteinase domain of MMP-26 were synthesized by Dr. Umesh Goli at the Biochemical Analysis, Synthesis and Sequencing Services Laboratory of the Department of Chemistry and Biochemistry, Florida State University (Tallahassee, FL). The sequence selected from the pro-domain was Thr50-Gln-Glu-Thr-Gln-Thr-Gln-Leu-Leu-Gln-Gln-Phe-His-Arg-Asn-Gly-Thr-Asp67, and the sequence selected from the metalloproteinase domain was Asp188-Lys-Asn-Glu-His-Trp-Ser-Ala-Ser-Asp-Thr-Gly-Tyr-Asn201of the prepro-enzyme. Using the BLAST search method at the National Center for Biotechnology Information web site against all of the sequences in the data banks, no peptide with >45% level of identity was found (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), predicting the antibodies directed against these two peptides should be specific. The purity of these peptides was verified by reverse-phase high performance liquid chromatography and mass spectrometry. Rabbit anti-human antibodies were then generated, purified, and characterized as described previously (19Zhao Y.G. Wei P. Sang Q.-X. Biochem. Biophys. Res. Commun. 2001; 289: 288-294Crossref PubMed Scopus (15) Google Scholar, 21Li H. Bauzon D.E. Xu X. Tschesche H. Cao J. Sang Q.-X. Mol. Carcinog. 1998; 22: 84-94Crossref PubMed Scopus (51) Google Scholar). Western blot analyses have demonstrated that these two antibodies are highly specific for MMP-26 because they do not cross-react with human matrilysin (MMP-7), stromelysin (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), and some other proteins tested (data not shown). Western blotting for MMP-26 was performed by lysing the cells with Tris-buffered saline (50 mm Tris and 150 mm NaCl, pH 7.4) containing 1.5% (v/v) Triton X-114 as described previously (21Li H. Bauzon D.E. Xu X. Tschesche H. Cao J. Sang Q.-X. Mol. Carcinog. 1998; 22: 84-94Crossref PubMed Scopus (51) Google Scholar). Aliquots (20 μl) of cell lysate and media containing equal volumes (20 μl) from each treatment treated with SDS sample buffer were then loaded onto an SDS-polyacrylamide gel. Samples were electrophoresed and then electroblotted onto a nitrocellulose membrane. Immunoreactive MMP-26 bands were visualized using a horseradish peroxidase or alkaline phosphatase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). Western blot analysis for MMP-9 was performed with a 1 μg/ml dilution of polyclonal anti-MMP-9 antibody (Oncogene Science, Cambridge, MA). MMP-9 bands were visualized using an alkaline phosphatase-conjugated secondary antibody (Jackson ImmunoResearch) followed by the addition of 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium. The blot membranes were then scanned, and the signal intensities were measured by integrated morphometry analysis (IMA) (Metamorph System, version 4.6r8, Universal Imaging Corporation, Inc., West Chester, PA). The signal intensities obtained were expressed as integrated optical density (the sum of the optical densities of all pixels that make up the object). All the bands used the same exclusive threshold for analysis. Cells were fixed in 50% methanol, 50% acetone for 15 min and permeated with 1% Triton X-100 in Tris-buffered saline for 15 min. Formalin-fixed paraffin-embedded human prostate cancer tissues were sectioned to 4 μm thickness and fixed on slides. The sections were dewaxed with xylene and rehydrated in 100 and 95% ethanol. Nonspecific antibody binding in cells and sections was blocked with blocking buffer (0.2% Triton X-100, 5% normal goat serum, and 3% bovine serum albumin in Tris-buffered saline) for 1 h at room temperature prior to overnight incubation with affinity-purified specific rabbit anti-human MMP-26 antibody in the same buffer (5 μg/ml for immunocytochemistry and 10 μg/ml for inmmunohistochemistry) or goat anti-human MMP-9 antibody (25 μg/ml for immunohistochemistry, R&D Systems, Minneapolis, MN) at 4 °C. Cells and sections were incubated with alkaline phosphatase-conjugated secondary antibody (Jackson ImmunoResearch) diluted (1:5000) in the blocking buffer for 4 h at room temperature. The signals were detected by adding Fast-Red (Sigma). Purified preimmune IgGs from the same animal were used as negative controls for MMP-26. Normal goat serum was used as a negative control for MMP-9. The sections were counterstained lightly with hematoxylin for viewing negatively stained cells. Full-length cDNA ofMMP-26 was amplified by PCR according to published sequences (6Park H.I. Ni J. Gerkema F.E. Liu D. Belozerov V.E. Sang Q.-X. J. Biol. Chem. 2000; 275: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and cloned into modified mammalian expression vector pCRTM3.1-Uni with a FLAG tag at its C-terminal as described (22Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). Following confirmation of cDNA sequencing, plasmids containing correct inserts were used as sense vectors and plasmids with reversibly inserted cDNA were used as antisense vectors (22Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). ARCaP cells were transfected with sense and antisense MMP-26cDNA-containing vectors using LipofectAMINE 2000 (Invitrogen) as described earlier (22Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar, 23Kang T. Zhao Y.-G. Pei D. Sucic J.F. Sang Q.-X. J. Biol. Chem. 2002; 277: 25583-25591Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Sense- and antisense-transfected cell lines were treated identically with regard to transfection conditions and maintenance in the selection medium. Stable transfectants were selected by growing the cells in 400 μg/ml Geneticin (G418; Invitrogen). Cells that survived were then expanded in the absence of G418 for additional studies. Stable transfectants were screened on the basis of FLAG and MMP-26 expression. Clones with MMP-26 sense- and antisense-integrated constructs were selected and analyzed for MMP-26 expression, invasive capabilities in modified Boyden chamber invasion assays, and co-localization with MMP-9. Parental ARCaP cells served as controls. The invasiveness of ARCaP cells cultured in the presence of MMP-26 or MMP-9 functional blocking antibodies, parental ARCaP cells, sense MMP-26- and antisense MMP-26-transfected cells through reconstructed ECM was determined as per our previous report (24Sang Q.-X. Jia M.-C. Schwartz M.A. Jaye M.C. Kleinman H.K. Ghaffari M.A. Luo Y.-L. Biochem. Biophys. Res. Commun. 2000; 274: 780-786Crossref PubMed Scopus (16) Google Scholar). The final concentrations of MMP-26 antibody were 10 and 50 μg/ml. The preimmune IgG from the same animal was used as control for MMP-26 antibody, and the final concentration was 50 μg/ml. The mouse anti-human MMP-9 monoclonal antibody is Ab-1, clone 6-6B, which is a functional neutralizing antibody that inhibits the enzymatic activity of MMP-9 (25Ramos-DeSimone N. Moll U.M. Quigley J.P. French D.L. Hybridoma. 1993; 12: 349-363Crossref PubMed Scopus (66) Google Scholar) (Oncogene Research Products, CalBiochem, La Jolla, CA). The final concentrations of MMP-9 monoclonal antibody were 10 and 25 μg/ml. The preimmune mouse IgG (Alpha Diagnostic Intl. Inc., San Antonio, TX) was used as control, and the concentration was 25 μg/ml. Briefly, modified Boyden chambers containing polycarbonate filters with 8-μm pores (Becton Dickinson, Boston, MA) were coated with 0.5 mg/ml human plasma FN (Invitrogen) or 0.5 mg/ml type IV collagen (Sigma). Three-hundred μl of prepared cell suspension (1 × 106 cells/ml) in serum-free medium was added to each insert, and 500 μl of media containing 10% fetal bovine serum was added to the lower chamber. After 60 h of incubation, invasive cells that had passed through the filters to the lower surface of the membrane were fixed in 4% paraformaldehyde (Sigma). The cells were then stained with 0.1% crystal violet solution and photographed with an Olympus DP10 digital camera (Melville, NY) under a Nikon FX microscope (Melville, NY). The cells were then counted by IMA. For statistical analyses, the number of invasive cells treated with preimmune IgG was assumed to reflect 100% cell invasion. The ratio of the number of invaded cells that were treated with antibody or theMMP-26 gene-transfected cells to preimmune IgG or parental cells, respectively, was used for subsequent comparative analyses by analysis of variance (ANOVA). Media from each insert was collected for Western blot and gelatin zymogram analyses. Cells were cultured on 8-well slides for 24 h, then fixed in fresh 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% Triton X-100 in 10% normal goat serum in phosphate-buffered saline. The fixed, permeabilized cells were stained for 1 h at room temperature with anti-human MMP-26 (25 μg/ml) or a goat anti-human antibody targeting MMP-9 (R&D Systems, Minneapolis, MN) (1:200 dilution). Secondary rhodamine red-X-conjugated mouse anti-rabbit IgG for MMP-26 or fluorescein-conjugated donkey anti-goat IgG (Jackson ImmunoResearch) for MMP-9 were subsequently applied at a 1:200 dilution for 1 h at room temperature. Slow Fade mounting medium was added to the slides, and fluorescence was analyzed using a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss, Germany) equipped with a multiphoton laser according to our previous report (23Kang T. Zhao Y.-G. Pei D. Sucic J.F. Sang Q.-X. J. Biol. Chem. 2002; 277: 25583-25591Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Images were processed for reproduction using Photoshop software version 6.0 (Adobe Systems, Mountainview, CA). Purified preimmune IgGs from the same animal were used as negative controls for MMP-26, and normal goat serum was used as a negative control for MMP-9. Samples were simultaneously stained with antibody and preimmune IgG on the same slide, and the areas of MMP-26 immunostaining were quantified by IMA. Four photographs were taken from each sample with an Olympus DP10 digital camera under a Nikon FX microscope. An appropriate color threshold was determined (color model, HSI; hue, 230–255; saturation and intensity, full spectrum), the glandular epithelia from each image was isolated into closed regions, and all areas of staining in compliance with these specific parameters were measured by IMA. The total area of these closed regions was determined by region measurement, and the ratio of signal area to total area was then determined. The average of the four ratios obtained from each sample was then used for subsequent analysis. The same color threshold was maintained for all samples. The preimmune staining ratio was subtracted from the antibody-staining ratio, and this value was then divided by the preimmune staining ratio to yield the reduced signal to background ratios used for subsequent comparative analyses by ANOVA. Statistical analysis of all samples was performed with the least significant difference correction of ANOVA for multiple comparisons. Data represent the mean ± S.D. from three experiments where differences withp < 0.05 were considered to be significant. Gelatin zymography was utilized for determination of MMP-9 activity levels following cleavage by MMP-26. Zymography revealed that pro-MMP-9 presented as 225-, 125-, and 94-kDa gelatinolytic bands under non-reducing conditions (Fig.1, A, lane 1, andB, lanes 1 and 6). The 225-kDa band is a homodimer of pro-MMP-9, the 125-kDa band is a heterodimer of pro-MMP-9 and neutrophil gelatinase-associated lipocalin, and the 94-kDa band is a monomer of pro-MMP-9 (17Sang Q.-X. Birkedal-Hansen H. Van Wart H.E. Biochim. Biophys. Acta. 1995; 1251: 99-108Crossref PubMed Scopus (95) Google Scholar, 26Tschesche H. Zolzer V. Triebel S. Bartsch S. Eur. J. Biochem. 2001; 268: 1918-1928Crossref PubMed Scopus (101) Google Scholar, 27Yan L. Borregaard N. Kjeldsen L. Moses M.A. J. Biol. Chem. 2001; 276: 37258-37265Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). New 215-, 115-, and 86-kDa bands were generated after incubation with MMP-26 (Fig. 1,A and B), and their activities were increased in a dose- and time-dependent manner (Fig. 1, A andB). Compared with MMP-7, the cleavage products generated by MMP-26 at the concentrations tested appear more stable (Fig. 1,A and B). However, pro-MMP-2 was not activated after incubation with identical concentrations of MMP-26 (data not shown). MMP-26 cleaved pro-MMP-9 (94 kDa) to yield a new 86-kDa band on a silver-stained gel under reducing conditions (Fig. 1C,lane 4). N-terminal sequencing showed that the 86-kDa protein had the sequence of MRTPRXG, which is the same N terminus as reported during activation of pro-MMP-9 by HgCl2 (28Triebel S. Blaser J. Reinke H. Knauper V. Tschesche H. FEBS Lett. 1992; 298: 280-284Crossref PubMed Scopus (28) Google Scholar), human fibroblast-type collagenase (MMP-1) (17Sang Q.-X. Birkedal-Hansen H. Van Wart H.E. Biochim. Biophys. Acta. 1995; 1251: 99-108Crossref PubMed Scopus (95) Google Scholar), phenylmercuric acid (29Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Sch
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