Secreted Versus Membrane-anchored Collagenases
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.002808
ISSN1083-351X
AutoresFarideh Sabeh, Xiaoyan Li, Thomas L. Saunders, R. Grant Rowe, Stephen J. Weiss,
Tópico(s)Connective tissue disorders research
ResumoFibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13−/−, Mmp8−/−, Mmp2−/−, Mmp9−/−, Mmp14−/− and Mmp16−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14−/− or Mmp16−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype. Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13−/−, Mmp8−/−, Mmp2−/−, Mmp9−/−, Mmp14−/− and Mmp16−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14−/− or Mmp16−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype. In the postnatal state, fibroblasts are normally embedded in a self-generated three-dimensional connective tissue matrix composed largely of type I collagen, the major extracellular protein found in mammals (1Sweeney S.M. Orgel J.P. Fertala A. McAuliffe J.D. Turner K.R. Di Lullo G.A. Chen S. Antipova O. Perumal S. Ala-Kokko L. Forlino A. Cabral W.A. Barnes A.M. Marini J.C. San Antonio J.D. J. Biol. Chem. 2008; 283: 21187-21197Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 2Eckes B. Zigrino P. Kessler D. Holtkötter O. Shephard P. Mauch C. Krieg T. Matrix Biol. 2000; 19: 325-332Crossref PubMed Scopus (201) Google Scholar, 3Grinnell F. Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Type I collagen not only acts as a structural scaffolding for the associated mesenchymal cell populations but also regulates gene expression and cell function through its interactions with collagen binding integrins and discoidin receptors (2Eckes B. Zigrino P. Kessler D. Holtkötter O. Shephard P. Mauch C. Krieg T. Matrix Biol. 2000; 19: 325-332Crossref PubMed Scopus (201) Google Scholar, 4Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar). Consistent with the central role that type I collagen plays in defining the structure and function of the extracellular matrix, the triple-helical molecule is resistant to almost all forms of proteolytic attack and can display a decades-long half-life in vivo (4Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar, 5Bank R.A. TeKoppele J.M. Oostingh G. Hazleman B.L. Riley G.P. Ann. Rheum. Dis. 1999; 58: 35-41Crossref PubMed Scopus (103) Google Scholar, 6Perumal S. Antipova O. Orgel J.P. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2824-2829Crossref PubMed Scopus (270) Google Scholar). Nonetheless, fibroblasts actively remodel type I collagen during wound healing, inflammation, or neoplastic states (2Eckes B. Zigrino P. Kessler D. Holtkötter O. Shephard P. Mauch C. Krieg T. Matrix Biol. 2000; 19: 325-332Crossref PubMed Scopus (201) Google Scholar, 7Costea D.E. Kulasekara K. Neppelberg E. Johannessen A.C. Vintermyr O.K. Am. J. Pathol. 2006; 168: 1889-1897Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 8Liu X. Wu H. Byrne M. Jeffrey J. Krane S. Jaenisch R. J. Cell Biol. 1995; 130: 227-237Crossref PubMed Scopus (244) Google Scholar, 9Johnsen M. Lund L.R. Rømer J. Almholt K. Danø K. Curr. Opin. Cell Biol. 1998; 10: 667-671Crossref PubMed Scopus (328) Google Scholar, 10Singer A.J. Clark R.A. N. Engl. J. Med. 1999; 341: 738-746Crossref PubMed Scopus (4738) Google Scholar, 11Arora P.D. Manolson M.F. Downey G.P. Sodek J. McCulloch C.A. J. Biol. Chem. 2000; 275: 35432-35441Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 12Crawford Y. Kasman I. Yu L. Zhong C. Wu X. Modrusan Z. Kaminker J. Ferrara N. Cancer Cell. 2009; 15: 21-34Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 13Page-McCaw A. Ewald A.J. Werb Z. Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (2224) Google Scholar). To date type I collagenolytic activity is largely confined to a small subset of fewer than 10 proteases belonging to either the cysteine proteinase or matrix metalloproteinase (MMP) 2The abbreviations used are: MMPmatrix metalloproteinaseRTreverse transcriptionFCSfetal calf serumPDGFplatelet-derived growth factorBrdUrdbromodeoxyuridine. gene families (4Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar, 14Creemers L.B. Hoeben K.A. Jansen D.C. Buttle D.J. Beertsen W. Everts V. Matrix Biol. 1998; 16: 575-584Crossref PubMed Google Scholar, 15Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar, 16Li Z. Hou W.S. Escalante-Torres C.R. Gelb B.D. Bromme D. J. Biol. Chem. 2002; 277: 28669-28676Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 17Ghersi G. Dong H. Goldstein L.A. Yeh Y. Hakkinen L. Larjava H.S. Chen W.T. J. Biol. Chem. 2002; 277: 29231-29241Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 18Li Z. Yasuda Y. Li W. Bogyo M. Katz N. Gordon R.E. Fields G.B. Brömme D. J. Biol. Chem. 2004; 279: 5470-5479Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). As all collagenases are synthesized as inactive zymogens, complex proteolytic cascades involving serine, cysteine, metallo, and aspartyl proteinases have also been linked to collagen turnover by virtue of their ability to mediate the processing of the pro-collagenases to their active forms (13Page-McCaw A. Ewald A.J. Werb Z. Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (2224) Google Scholar, 15Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar, 19Ra H.J. Parks W.C. Matrix Biol. 2007; 26: 587-596Crossref PubMed Scopus (459) Google Scholar). After activation, each collagenase can then cleave native collagen within its triple-helical domain, thus precipitating the unwinding or "melting" of the resulting collagen fragments at physiologic temperatures (4Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar, 15Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar). In turn, the denatured products (termed gelatin) are susceptible to further proteolysis by a broader class of "gelatinases" (4Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar, 15Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar). Collagen fragments are then either internalized after binding to specific receptors on the cell surface or degraded to smaller peptides with potent biological activity (20Madsen D.H. Engelholm L.H. Ingvarsen S. Hillig T. Wagenaar-Miller R.A. Kjøller L. Gårdsvoll H. Høyer-Hansen G. Holmbeck K. Bugge T.H. Behrendt N. J. Biol. Chem. 2007; 282: 27037-27045Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 21Zhang Y. Zhou Z.H. Bugge T.H. Wahl L.M. J. Immunol. 2007; 179: 3297-3304Crossref PubMed Scopus (34) Google Scholar, 22Gaggar A. Jackson P.L. Noerager B.D. O'Reilly P.J. McQuaid D.B. Rowe S.M. Clancy J.P. Blalock J.E. J. Immunol. 2008; 180: 5662-5669Crossref PubMed Scopus (262) Google Scholar, 23O'Reilly P.J. Gaggar A. Blalock J.E. Curr. Opin. Pharmacol. 2008; 8: 242-248Crossref PubMed Scopus (22) Google Scholar, 24Lin M. Jackson P. Tester A.M. Diaconu E. Overall C.M. Blalock J.E. Pearlman E. Am. J. Pathol. 2008; 173: 144-153Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). matrix metalloproteinase reverse transcription fetal calf serum platelet-derived growth factor bromodeoxyuridine. Previous studies by our group as well as others have identified MMPs as the primary effectors of fibroblast-mediated collagenolysis (20Madsen D.H. Engelholm L.H. Ingvarsen S. Hillig T. Wagenaar-Miller R.A. Kjøller L. Gårdsvoll H. Høyer-Hansen G. Holmbeck K. Bugge T.H. Behrendt N. J. Biol. Chem. 2007; 282: 27037-27045Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (490) Google Scholar, 26Lee H. Overall C.M. McCulloch C.A. Sodek J. Mol. Biol. Cell. 2006; 17: 4812-4826Crossref PubMed Scopus (90) Google Scholar). Interestingly, adult mouse fibroblasts express at least six MMPs that can potentially degrade type I collagen, raising the possibility of multiple compensatory networks that are designed to preserve collagenolytic activity (25Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (490) Google Scholar). Four of these collagenases belong to the family of secreted MMPs, i.e. MMP-13, MMP-8, MMP-2, and MMP-9, whereas the other two enzymes are members of the membrane-type MMP subgroup, i.e. MMP-14 (MT1-MMP) and MMP-16 (MT3-MMP) (13Page-McCaw A. Ewald A.J. Werb Z. Nat. Rev. Mol. Cell Biol. 2007; 8: 221-233Crossref PubMed Scopus (2224) Google Scholar, 27Shi J. Son M.Y. Yamada S. Szabova L. Kahan S. Chrysovergis K. Wolf L. Surmak A. Holmbeck K. Dev. Biol. 2008; 313: 196-209Crossref PubMed Scopus (87) Google Scholar, 28Bigg H.F. Rowan A.D. Barker M.D. Cawston T.E. FEBS J. 2007; 274: 1246-1255Crossref PubMed Scopus (96) Google Scholar, 29Gioia M. Monaco S. Fasciglione G.F. Coletti A. Modesti A. Marini S. Coletta M. J. Mol. Biol. 2007; 368: 1101-1113Crossref PubMed Scopus (64) Google Scholar). From a functional perspective, the specific roles that can be assigned to secreted versus membrane-anchored collagenases remain undefined. As such, fibroblasts were isolated from either wild-type mice or mice harboring loss-of-function deletions in each of the major secreted and membrane-anchored collagenolytic genes, and the ability of the cells to degrade type I collagen was assessed. Herein, we demonstrate that fibroblasts mobilize either secreted or membrane-anchored MMPs to effectively degrade type I collagen in qualitatively and quantitatively distinct fashions. However, under conditions where fibroblasts use either secreted and membrane-anchored MMPs to exert quantitatively equivalent collagenolytic activity, only MT1-MMP plays a required role in supporting a collagen-invasive phenotype. These data establish a new paradigm wherein secreted collagenases are functionally limited to bulk collagenolytic processes, whereas MT1-MMP uniquely arms the fibroblast with a focalized degradative activity that mediates subjacent collagenolysis as well as invasion. Mice with targeted deletions in Mmp14, Mmp2, Mmp9, Mmp8, Mmp13, Mmp16, or Timp2 have been described previously (25Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (490) Google Scholar, 27Shi J. Son M.Y. Yamada S. Szabova L. Kahan S. Chrysovergis K. Wolf L. Surmak A. Holmbeck K. Dev. Biol. 2008; 313: 196-209Crossref PubMed Scopus (87) Google Scholar, 30Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res. 1998; 58: 1048-1051PubMed Google Scholar, 31Liu Z. Shipley J.M. Vu T.H. Zhou X. Diaz L.A. Werb Z. Senior R.M. J. Exp. Med. 1998; 188: 475-482Crossref PubMed Scopus (213) Google Scholar, 32Balbín M. Fueyo A. Knäuper V. López J.M. Alvarez J. Sánchez L.M. Quesada V. Bordallo J. Murphy G. López-Otín C. J. Biol. Chem. 2001; 276: 10253-10262Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 33Inada M. Wang Y. Byrne M.H. Rahman M.U. Miyaura C. López-Otín C. Krane S.M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 17192-17197Crossref PubMed Scopus (455) Google Scholar, 34Wang Z. Juttermann R. Soloway P.D. J. Biol. Chem. 2000; 275: 26411-26415Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Mmp16 (GenBankTM accession number AB021228) was disrupted by replacing sequences from base 814 to base 821 within exon 5 with a LacZ-Neo cassette to generate the Mmp16tmlDgen allele. Frozen embryos were obtained from the Mutant Mouse Regional Resource Center at the University of North Carolina at Chapel Hill. Mmp16 homozygous knock-out mice are maintained on a C57BL/6 background. Mmp16 expression was assessed by RT-PCR using forward primers for mouse Mmp16, 5′-GGAGACAGTTCCCCATTTGA-3′, and reverse primers, 5′-CGTTGGAATGTTCCAGTCCT-3′. Fibroblasts were isolated from the dorsal skin of 2–8-week-old mice (passage 2–8) and were cultured in Dulbecco's modified Eagle's medium supplemented with either 10% heat-inactivated fetal calf serum (FCS; Atlanta Biologicals) or heat-inactivated mouse serum (from control, Mmp2−/− or Mmp9−/− mice), 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml Fungizone, and 2 mml-glutamine. Cells were cultured on type I collagen matrix for 2 days in serum-free media in the absence and presence of plasminogen, and total RNA was isolated using TRIzol reagent (Invitrogen). RT-PCR was performed with 1 μg of total RNA and 10 μm concentrations of specific primers (25Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (490) Google Scholar, 36Hotary K.B. Yana I. Sabeh F. Li X.Y. Holmbeck K. Birkedal-Hansen H. Allen E.D. Hiraoka N. Weiss S.J. J. Exp. Med. 2002; 195: 295-308Crossref PubMed Scopus (181) Google Scholar) using the One-Step RT-PCR System reagent (Invitrogen). Full-length MT1-MMP and deletion mutants (37Li X.Y. Ota I. Yana I. Sabeh F. Weiss S.J. Mol. Biol. Cell. 2008; 19: 3221-3233Crossref PubMed Scopus (100) Google Scholar) were subcloned into the pBMN-Z retroviral vector (Addgene, Cambridge, MA). Retroviral vectors were co-transfected into HEK293 cells with ecotropic packaging plasmids using Lipofectamine 2000 (Invitrogen). Six hours post-transfection, cells were seeded at low density, and retroviral supernatants were collected at 48 h. Supernatants were diluted 1:1 with fibroblast culture medium supplemented with 6 μg/ml Polybrene (Sigma) and added to dermal fibroblast cultures. Cells were infected for 48 h. Type I collagen degradation was evaluated using a modification of a previously described method (38Netzel-Arnett S. Mitola D.J. Yamada S.S. Chrysovergis K. Holmbeck K. Birkedal-Hansen H. Bugge T.H. J. Biol. Chem. 2002; 277: 45154-45161Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In brief, 24-well plates were coated with collagen gel (100 μg/well), and 5 × 104 fibroblasts in 35 μl of medium were seeded in the center of each well and allowed to adhere for 8 h. After washing, 0.5 ml of serum-free medium was added to each well with or without PDGF-BB (10 ng/ml), plasminogen (20 μg/ml), or protease inhibitors as indicated. After 5 days, cells were removed by trypsin/EDTA or detergent lysis, and the remaining collagen film was stained with Coomassie Brilliant Blue. Alternatively, collagen degradation products were quantified by hydroxyproline release into the conditioned medium after an ethanol precipitation step (70% v/v) as described (39Creemers L.B. Jansen D.C. van Veen-Reurings A. van den Bos T. Everts V. Biotechniques. 1997; 22: 656-658Crossref PubMed Scopus (125) Google Scholar). Serum-free media were supplemented with recombinant tissue inhibitor of metalloproteinases-1 or -2 (TIMP-1; 7.5 μg/ml or TIMP-2; 2.5 μg/ml; Fuji Chemical Industries), the synthetic MMP inhibitor, BB-94 (5 μm final concentration in 0.1% DMSO; British Biotechnology, Oxford, UK), the cysteine proteinase inhibitor, E-64d (100 μm; Sigma), the aspartyl proteinase inhibitor, pepstatin A (50 μm; Sigma), or the serine proteinase inhibitors, soybean trypsin inhibitor, SBTI (100 μg/ml), or aprotinin (200 μg/ml; Roche Applied Science). Type I collagen was acid- or pepsin- extracted from rat or mice tail tendons (4Birkedal-Hansen H. Methods Enzymol. 1987; 144: 140-171Crossref PubMed Scopus (130) Google Scholar, 40Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (512) Google Scholar). Collagen gels were prepared in 24-mm Transwell dishes (3-μm pore size; Corning, Inc.) at a final concentration of 2.2 mg/ml (40Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (512) Google Scholar). After gelling (45 min at 37 °C), 1.5–2 × 105 cells in plasminogen-free or plasminogen-supplemented (20 μg/ml) media were added to the upper compartment of the Transwell dishes, and PDGF-BB (10 ng/ml) was added to the lower compartment of the Transwell chambers to initiate fibroblast invasion. Invasive activity was visualized by phase contrast microscopy. The number of invading cells was quantified as the mean ± 1 S.E. of at least three experiments as described (36Hotary K.B. Yana I. Sabeh F. Li X.Y. Holmbeck K. Birkedal-Hansen H. Allen E.D. Hiraoka N. Weiss S.J. J. Exp. Med. 2002; 195: 295-308Crossref PubMed Scopus (181) Google Scholar). Fibroblast motility was monitored by culturing 1 × 105 cells atop collagen gels in a 35-μl droplets. After attachment, the loose cells were washed away, and serum-free medium was supplemented with 10% FCS added to initiate a motile response. After 48 h, the cells were stained with toluidine blue. The distance migrated by the advancing front of cells (i.e. 3 or more cells) from the confluent area compared with 0 days was measured in 5 randomly selected fields. To monitor BrdUrd uptake in proliferating cells, fibroblasts in two-dimensional culture were cultured in 0.5% FCS overnight. Fresh media were then added containing either 0.5% FCS (control), 0.5% FCS supplemented with 10 ng/ml fibroblast growth factor-2, or 10% FCS. After overnight incubation, 10 μm BrdUrd was added for 60 min, after which time the cultures were washed with PBS, fixed, and processed with anti-BrdUrd antibody (Roche Applied Science). The cells were counterstained with propidium iodide (Invitrogen) to determine total cell number. The BrdUrd-positive cells were counted in 10 randomly selected fields and expressed as percent of total cells (mean ± S.E.; n = 3). The fluorescence images were captured using Spot digital camera (Diagnostic Instruments, Inc.) through a Leica upright microscope. Collagen cultures were prepared for light microscopy as described (25Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (490) Google Scholar, 40Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (512) Google Scholar), and sections (5–7 μm thick) were stained with hematoxylin and eosin. For electron microscopy, gels were fixed in 2% glutaraldehyde, 1.5% paraformaldehyde in 0.1 m sodium cacodylate buffer and processed as described (40Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (512) Google Scholar, 41Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. Cell. 1998; 95: 365-377Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar). When a concentric island of mouse fibroblasts is established on the surface of a circular, three-dimensional gel of type I collagen and stimulated with PDGF-BB under plasminogen-free conditions, the fibroblasts display increased collagenolytic activity that is confined to the substratum directly subjacent to the area covered by the seeded inoculum of cells (i.e. sites of collagen degradation are visualized as zones of clearing where the underlying gel is no longer observed after staining with Coomassie Blue and de-staining; see Fig. 1A). Although multiple classes of proteolytic enzymes have been linked indirectly to type I collagen degradation (14Creemers L.B. Hoeben K.A. Jansen D.C. Buttle D.J. Beertsen W. Everts V. Matrix Biol. 1998; 16: 575-584Crossref PubMed Google Scholar, 42Cox S.W. Eley B.M. Kiili M. Asikainen A. Tervahartiala T. Sorsa T. Oral Dis. 2006; 12: 34-40Crossref PubMed Scopus (53) Google Scholar), fibroblasts stimulated with PDGF-BB in the presence of cysteine (E-64d), aspartyl (pepstatin A), or serine (SBTI or aprotinin) proteinase inhibitors maintain collagenolytic activity as assessed by Coomassie Blue staining or the solubilization of hydroxyproline-containing collagen fragments (Fig. 1, A and B). By contrast, the synthetic MMP inhibitor, BB-94, completely blocks collagenolysis (Fig. 1, A and B). Although fibroblasts focus collagenolytic activity to the cell-matrix interface under plasminogen-free conditions, these cells can also process plasminogen to plasmin, a potent direct- or indirect- activator of secreted MMP zymogens (43Almholt K. Juncker-Jensen A. Green K.A. Solberg H. Lund L.R. Romer J. Edwards D. Hoyer-Hansen G. Blasi F. Sloane B.F. The Cancer Degradome. Springer Science, New York2008: 203-222Crossref Scopus (3) Google Scholar, 44Green K.A. Almholt K. Ploug M. Rønø B. Castellino F.J. Johnsen M. Bugge T.H. Rømer J. Lund L.R. J. Invest. Dermatol. 2008; 128: 2092-2101Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To determine whether collagen degradation by fibroblasts can be augmented by this serine proteinase axis, PDGF-BB-stimulated cells were cultured atop collagen gels in the presence of plasminogen. Under these conditions plasminogen is processed to plasmin (3.3 ± 0.2 munits/106 cells/24 h) via a plasminogen activator-dependent process (data not shown), which increases collagen degradation 2.5-fold (Fig. 1, A and B). Coincident with the enhancement of collagenolytic activity, the zone of collagen dissolution is extended beyond the confines of the fibroblast island to include the entire surface of the culture dish (Fig. 1, A and B). Although neither E-64d nor pepstatin A affected plasminogen-dependent collagenolysis, the plasmin inhibitor, aprotinin, specifically blocks that portion of the collagen degradation that occurs at sites distant from the seeded cells (herein referred to as "bulk" collagenolysis as opposed to subjacent collagen degradation) while leaving subjacent collagenolysis unaffected (Fig. 1, A and B). Consistent with the conclusion that the enhanced levels of fibroblast-mediated collagen degradation observed in the presence of plasminogen remains dependent on the activity of collagenolytic MMPs, all degradative activity is quenched in the presence of BB-94 (Fig. 1, A and B) without affecting plasmin activity (data not shown). The bulk dissolution of the collagen film observed in the presence of plasminogen does not occur as a consequence of fibroblasts moving from the centrally located inoculation site as the area of collagen film covered by the cells is similar in the absence or presence of either plasminogen or BB-94 (Fig. 1C). Furthermore, the addition of plasminogen does not alter the pattern of Mmp or Timp2 expression (Fig. 1D). During matrix remodeling events in vivo, fibroblasts express a family of MMP zymogens which potentially participate in collagen degradation by either functioning as direct-acting collagenolysins, promoting MMP activation, or accelerating the further degradation of collagen cleavage fragments (45Hanemaaijer R. Sorsa T. Konttinen Y.T. Ding Y. Sutinen M. Visser H. van Hinsbergh V.W. Helaakoski T. Kainulainen T. Rönkä H. Tschesche H. Salo T. J. Biol. Chem. 1997; 272: 31504-31509Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 46Okada A. Tomasetto C. Lutz Y. Bellocq J.P. Rio M.C. Basset P. J. Cell Biol. 1997; 137: 67-77Crossref PubMed Scopus (193) Google Scholar, 47Mohan R. Rinehart W.B. Bargagna-Mohan P. Fini M.E. J. Biol. Chem. 1998; 273: 25903-25914Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 48Yamamoto H. Flannery M.L. Kupriyanov S. Pearce J. McKercher S.R. Henkel G.W. Maki R.A. Werb Z. Oshima R.G. Genes Dev. 1998; 12: 1315-1326Crossref PubMed Scopus (255) Google Scholar, 49Wu N. Opalenik S. Liu J. Jansen E.D. Giro M.G. Davidson J.M. Matrix Biol. 2002; 21: 149-161Crossref PubMed Scopus (33) Google Scholar). As previously described (25Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (490) Google Scholar), fibroblasts stimulated with PDGF-BB atop a collagen substratum express Mmp13, Mmp8, Mmp2, Mmp9, Mmp14, and Mmp16 (Fig. 1D). To identify the individual MMPs, or MMP cascades, that drive the collagenolytic phenotype under plasminogen-free conditions, fibroblasts were isolated from each of the respective null mice, and the ability of resting or PDGF-BB-stimulated cells to degrade type I collagen was assessed. Despite the fact that MMP-13, MMP-8, MMP-2, and MMP-9 can each display collagenolytic activity under cell-free conditions (15Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar, 28Bigg H.F. Rowan A.D. Barker M.D. Cawston T.E. FEBS J. 2007; 274: 1246-1255Crossref PubMed Scopus (96) Google Scholar, 29Gioia M. Monaco S. Fasciglione G.F. Coletti A. Modesti A. Marini S. Coletta M. J. Mol. Biol. 2007; 368: 1101-1113Crossref PubMed Scopus (64) Google Scholar), fibroblasts null for any single member of the secreted collagenases degrade collagen comparably to control cells in either the absence or presence of PDGF-BB (Fig. 2, A and B). By contrast, MT1-MMP-deficient fibroblasts display a complete loss of subjacent collagenolytic activity under plasminogen-free conditions (Fig. 2, A and B) while maintaining wild-type levels of Mmp13, Mmp8, Mmp2, Mmp9, and Mmp16 (data not shown). Although MT3-MMP has recently been described as a type I collagenolysin (27Shi J. Son M.Y. Yamada S. Szabova L. Kahan S. Chrysovergis K. Wolf L. Surmak A. Holmbeck K. Dev. Biol. 2008; 313: 196-209Crossref PubMed Scopus (87) Google Scholar), the membrane-anchored enzyme does not play a key role in fibroblast-dependent collagen degradation as the null cells retain full collagenolytic activity (Fig. 2, A and B) without displaying compensatory changes in Mmp14 expression (data not shown). Although endogenously derived TIMP-2 can support MT1-MMP-dependent MMP-2 activation (15Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (970) Google Scholar, 50Murphy G. Gavrilovic J. Curr. Opin. Cell Biol. 1999; 11: 614-621Crossref PubMed Scopus (345) Google Scholar, 51Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (420) Google Scholar), Timp2−/− fibroblasts mount normal collagenolytic responses (Fig. 2, A and B). In each of the null populations studied, save for Mmp14−/− cells, subjacent degradation by fibroblasts remains insensitive to TIMP-1, an end
Referência(s)