Functional Interplay between Type I Collagen and Cell Surface Matrix Metalloproteinase Activity
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m005631200
ISSN1083-351X
AutoresShawn M. Ellerbroek, Yi Wu, Christopher M. Overall, M. Sharon Stack,
Tópico(s)Peptidase Inhibition and Analysis
ResumoType I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves β1 integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both α2β1 and α3β1 integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of α3β1 strongly promotes activation of pro-MMP-2, whereas α2β1integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance. Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves β1 integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both α2β1 and α3β1 integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of α3β1 strongly promotes activation of pro-MMP-2, whereas α2β1integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance. matrix metalloproteinase membrane type-1 matrix metalloproteinase tissue inhibitor of matrix metalloproteinase concanavalin A recombinant collagen binding domain recombinant carboxyl hemopexin domain phosphate-buffered saline Tris-buffered saline bovine serum albumin monoclonal antibody collagenase-resistant 4-morpholineethanesulfonic acid MMP inhibitor Chinese hamster ovary The MMP family is composed of at least 25 zinc-dependent extracellular endopeptidases whose activities are regulated predominantly by expression as inactive precursors, or zymogens (1Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar, 2Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar, 3Ellerbroek S.M. Stack M.S. BioEssays. 1999; 11: 940-949Crossref Scopus (133) Google Scholar). Although precise physiological activators of MMPs1 are unknown, a variety of serine proteinases and other MMPs in the extracellular milieu execute the initial propeptide cleavage eventsin vitro (2Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar). An exception to serine protease activation is pro-MMP-2 (72-kDa gelatinase A), which lacks the necessary basic amino acid cleavage sites in its pro-domain (4Corcoran M.L. Hewitt R.E. Kleiner Jr., D.E. Stetler-Stevenson W.G. Enzyme Protein. 1996; 49: 7-19Crossref PubMed Scopus (182) Google Scholar). A primary mechanism of pro-MMP-2 activation involves zymogen association with the cell surface via formation of a ternary complex containing tissue inhibitor of metalloproteinase (TIMP)-2 and membrane type 1-MMP (MT1-MMP, MMP-14) (3Ellerbroek S.M. Stack M.S. BioEssays. 1999; 11: 940-949Crossref Scopus (133) Google Scholar, 4Corcoran M.L. Hewitt R.E. Kleiner Jr., D.E. Stetler-Stevenson W.G. Enzyme Protein. 1996; 49: 7-19Crossref PubMed Scopus (182) Google Scholar, 5Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 6Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). Following trimeric complex formation, it is hypothesized that a neighboring MT1-MMP molecule that is not associated with TIMP-2 cleaves pro-MMP-2 at the Asn37–Leu38 peptide bond within the pro-domain (7Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Intermediately processed MMP-2 (Leu38–MMP-2) undergoes further concentration-dependent autolytic cleavage(s) to generate mature enzymes that can be released into the soluble phase or remain surface-associated (8Atkinson S.J. Crabbe T. Cowell S. Ward R.V. Butler M.J. Sato H. Seiki M. Reynolds J.J. Murphy G. J. Biol. Chem. 1995; 270: 30479-30485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Although biological mechanisms of active MMP-2 release from the cell surface are not well characterized and dissociation kinetics provide little insight, cellular binding affinities may shift following pro-MMP-2 cleavage.Culturing a variety of cell types within a three-dimensional gel of type I collagen stimulates cellular activation of pro-MMP-2 (9Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4544PubMed Google Scholar, 10Tomesek J.J. Halliday N.L. Updike D.L. Ahern-Moore J.S. Vu T.K. Liu R.W. Howard E.W. J. Biol. Chem. 1997; 272: 7482-7787Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Haas T.L. Davis S.J. Madri J.A. J. Biol. Chem. 1998; 273: 3604-3610Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar). Although MT1-MMP is implicated in MMP-2 processing, regulation of cellular events that promote MMP processing are poorly understood. As cellular interaction with type I collagen is mediated largely through integrin receptors, it has been postulated that collagen stimulation occurs either directly or indirectly through integrin signaling (12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar, 13Seltzer J.L. Lee A.Y. Akers K.T. Sudbeck B. Southon E.A. Wayner E.A. Eisen A.Z. Exp. Cell. Res. 1994; 213: 365-374Crossref PubMed Scopus (122) Google Scholar, 14Theret N. Lehti K. Musso O. Clement B. Hepatology. 1999; 30: 462-468Crossref PubMed Scopus (118) Google Scholar, 15Nguyen M. Arkell J. Jackson C.J. Int. J. Biochem. Cell Biol. 2000; 32: 621-631Crossref PubMed Scopus (41) Google Scholar, 16Boudreau N. Bissell M.J. Curr. Opin. Cell Biol. 1998; 10: 640-646Crossref PubMed Scopus (313) Google Scholar). In support, we have previously demonstrated that culturing DOV13 ovarian cancer cells in a three-dimensional collagen gel elicits a strong pro-MMP-2 activation response that can be mimicked by clustering of β1 integrin receptors (12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar). Furthermore, pro-MMP-2 activation coincides with the processing of MT1-MMP into truncated 55- and 43-kDa forms on the cell surface. In this study, we utilize a variety of approaches to elucidate the biochemical requirements of type I collagen stimulation of MMP zymogen activation, characterize processed forms of MT1-MMP that are generated in this response, and examine the proteinase requirements for cellular invasion of type I collagen gels. Our findings illustrate a general mechanism by which cells may regulate cell surface-associated MMP activity via interactions with pericellular collagen matrix.DISCUSSIONMT1-MMP is a cell surface activator of pro-MMP-2 and has been implicated in collagen invasion and turnover (33Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 34Cockett M.I. Murphy G. Birch B.L. O'Connell J.P. Crabbe T. Millican A.T. Hart I.R. Docherty A.J. Biochem. Soc. Symp. 1998; 63: 295-313PubMed Google Scholar, 35Chambers A.F. Matrisian L.M. J. Natl. Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1426) Google Scholar, 36Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (507) Google Scholar). In this study, DOV13 ovarian cancer cells activate MT1-MMP as a consequence of culture in type I collagen gels and display MMP-dependent invasion of type I collagen, indicating that MMP activity is required for removal of collagen matrix constraints during invasion. However, migration over two-dimensional collagen is not impeded by MMP inhibitors. Although TIMP-1 does not interact with MT1-MMP, TIMP-2 specifically binds the proteinase, functioning in both inhibition and stabilization of the enzyme on the cell surface (26Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 32Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The ability of exogenous TIMP-2, in contrast to TIMP-1, to inhibit DOV13 collagen gel invasion implicates a cell surface proteolytic cascade initiated by MT1-MMP. This is further supported by data demonstrating that inhibition of MMP-2 cellular activation or collagen binding using rCD and rCBD (17Steffensen B. Bigg H.F. Overall C.M. J. Biol. Chem. 1998; 273: 20622-20628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), respectively, has no effect on collagen invasion. As the cellular events that govern the collagen-induced MMP-2/MT1-MMP response are unclear and technically difficult to assess in three-dimensional collagen gel systems, a variety of biochemical approaches were employed in this study to dissect the interplay between collagen-cell interactions and regulation of cell surface MMP activity.DOV13 cells bind type I collagen via α2β1and α3β1 integrins. Recognition of collagen by these integrins depends on retention of the triple helical conformation, as thermal gelation of collagen abrogates cellular adhesion. Collagenase-cleaved type I collagen produces ¾ and ¼ fragments that display a lower T m than intact fibrils (37Danielsen C.C. Biochem. J. 1987; 247: 725-729Crossref PubMed Scopus (28) Google Scholar). Adhesion data in the current study demonstrate that the triple helical conformation of the collagen fragments is stabilized at low coating temperatures but is lost under physiological coating conditions (21Messent A.J. Tuckwell D.S. Knauper V. Humphries M.J. Murphy G. Gavrilovic J. J. Cell Sci. 1998; 111: 1127-1135Crossref PubMed Google Scholar). Together, these data suggest that pericellular type I collagenolysis will reduce α2β1 and α3β1 integrin-mediated cell-matrix contacts. By using a similar approach, the appearance of cryptic αVβ3 integrin-binding sites (RGD) in collagenase-generated collagen fragments was reported (21Messent A.J. Tuckwell D.S. Knauper V. Humphries M.J. Murphy G. Gavrilovic J. J. Cell Sci. 1998; 111: 1127-1135Crossref PubMed Google Scholar). However, DOV13 cells adhere weakly to type I gelatin or ¾ and ¼ fragments, and aggregation of αVβ3 integrins on the surface of DOV13 cells does not elicit a cellular MMP processing response (12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar, 20Moser T.L. Pizzo S.V. Bafetti L.M. Fishman D.A. Stack M.S. Int. J. Cancer. 1996; 67: 695-701Crossref PubMed Scopus (103) Google Scholar). Nevertheless, the exposure of cryptic αVβ3- or αVβ5-binding sites in collagenase-cleaved collagen may further influence MMP expression in a cell type-specific manner. In support of this observation, we have previously demonstrated that vitronectin-induced aggregation of melanoma cell αVβ3 integrins up-regulates MMP-2 expression (38Bafetti L.M. Young T.N. Itoh Y. Stack M.S. J. Biol. Chem. 1998; 273: 143-149Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Relative to collagen gel penetration, DOV13 cells rapidly invade a gelatin matrix. Although pro-MMP-2 activation is not up-regulated over basal levels under these conditions, additional proteinases of other mechanistic classes can provide gelatinase activity (1Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar, 39Pineiro-Sanchez M.L. Goldstein L.A. Dodt J. Howard L. Yeh Y. Chen W.T. J. Biol. Chem. 1997; 272: 7595-7601Crossref PubMed Scopus (124) Google Scholar, 40Andreasen P.A Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (834) Google Scholar). Together these data indicate that collagenase activity provided by MT1-MMP is critical to invasion of an intact collagen matrix. Subsequent clearance of resultant fragments can then proceed by activation of cell surface MMP-2 along with contributions from other cell surface proteinases including seprase (39Pineiro-Sanchez M.L. Goldstein L.A. Dodt J. Howard L. Yeh Y. Chen W.T. J. Biol. Chem. 1997; 272: 7595-7601Crossref PubMed Scopus (124) Google Scholar) and the components of the plasminogen activator/plasmin system (40Andreasen P.A Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (834) Google Scholar), which have been implicated in DOV13 cellular invasion of Matrigel (22Ellerbroek S.M. Hudson L.G. Stack M.S. Int. J. Cancer. 1998; 78: 331-337Crossref PubMed Scopus (74) Google Scholar).Stimulation of pro-MMP-2 activation does not require collagenolysis, as collagenase-resistant collagen is as efficacious as wild type type I collagen at inducing pro-MMP-2 processing. However, thermal denaturation of either collagen abolishes the ability to enhance MMP activation, suggesting that α2β1 and/or α3β1 integrin binding to intact triple-helical collagen mediates the MMP activation response in DOV13 cells. By using subunit-specific antibodies to dissect integrin requirements for MMP processing, our data demonstrate that clustering of α3 integrins promotes a stronger cellular MMP processing response than α2 integrin aggregation. A potential role for α integrin-specific regulation has been demonstrated previously for type I collagen-induced cellular responses, including those involving MMP-1 expression (41Langholz O. Rockel D. Mauch C. Kozlowska E. Bank I. Krieg T. Eckes B. J. Cell Biol. 1995; 131: 1903-1915Crossref PubMed Scopus (378) Google Scholar, 42Lichtner R.B. Howlett A.R. Lerch M. Xuan J.A. Brink J. Langton-Webster B. Schneider M.R. Exp. Cell Res. 1998; 240: 368-376Crossref PubMed Scopus (40) Google Scholar). Administration of function blocking antibodies against either α integrin subunit reveals a role for both receptors during invasion. Although this study implicates the α3β1 heterodimer in mediating the MT1-MMP response, it is likely that both α2β1 and α3β1integrins provide a mechanical advantage to the migration component of invasion. Furthermore, it is unclear at this level of investigation whether α2β1 integrins are required for effective dispersal of α3β1 into focal adhesions (43Grenz H. Carbonetto S. Goodman S.L. J. Cell Sci. 1993; 105: 739-751PubMed Google Scholar, 44DiPersio C.M. Shah S. Hynes R.O. J. Cell Sci. 1995; 108: 2321-2336Crossref PubMed Google Scholar). Interestingly, α3β1integrins have recently been hypothesized to play a major organizational role in the formation of invadopodia in response to cellular engagement of type I collagen (45Mueller S.C. Ghersi G. Akiyama S.K. Sang Q.X. Howard L. Pineiro-Sanchez M. Nakahara H. Yeh Y. Chen W.T. J. Biol. Chem. 1999; 274: 24947-24952Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). As MT1-MMP also localizes to invadopodia (27Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (360) Google Scholar, 28Urena J.M. Merlos-Suarez A. Baselga J. Arribas J. J. Cell Sci. 1999; 112: 773-784Crossref PubMed Google Scholar, 29Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. J. Biol. Chem. 2000; 275: 15006-15013Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), it is possible that α3β1 clustering in DOV13 cells selectively initiates cellular events that mimic formation of invadopodial projections, which in turn regulate MT1-MMP activity. Moreover, our data demonstrate localization of MT1-MMP immunoreactivity to the periphery of clustered β1 integrins, indicating that MT1-MMP redistribution occurs during integrin clustering events. This observation, together with previous reports of MT1-MMP localization to integrin-rich cellular protrusions, suggests a cellular regulatory mechanism for MT1-MMP aggregation, thereby promoting effective pro-MMP-2 processing and efficient matrix degradation. As MT1-MMP can function as a collagenase (31D'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Eur. J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (383) Google Scholar) and MT1-MMP null mice exhibit severe deficiencies in collagen remodeling (33Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar), localization of the enzyme to cellular collagen receptors could clearly influence physiologic events such as collagen gel contraction, adhesion, and invasion. In addition, pro-MMP-2 bound to intact peri-cellular collagen may readily infiltrate the MT1-MMP activation pathway, resulting in a switch from a collagenase to a gelatinase environment as pro-MMP-2 activation and collagen triple helix denaturation ensues (17Steffensen B. Bigg H.F. Overall C.M. J. Biol. Chem. 1998; 273: 20622-20628Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).It has recently been demonstrated that exogenous MT1-MMP overexpressed in a MMP-2 null background undergoes autolysis to a 43-kDa form, the rate of which is regulated by TIMP-2 (32Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Similarly, endogenously expressed MT1-MMP in DOV13 cells exists in two major forms of 55 and 43 kDa (12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar). The current data indicate that the 55-kDa form of MT1-MMP is the active TIMP-2-binding species, whereas the 43-kDa form is an inactive autolysis product. This result is consistent with the amino-terminal sequences of similar MT1-MMP species obtained from overexpression systems, which demonstrate a loss of essential amino acids in the zinc-binding consensus sequence (30Lehti K. Lohi J. Valtanen H. Keski-Oja J. Biochem. J. 1998; 334: 345-353Crossref PubMed Scopus (201) Google Scholar, 32Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Although catalytically inactive, the 43-kDa form of MT1-MMP is nevertheless retained on the cell surface. As this 43-kDa species contains the carboxyl-terminal domains necessary for invadopodial localization and enzyme aggregation, it is interesting to speculate that MT1-MMP-mediated proteolysis may be down-regulated through the dilution of active enzyme with truncated proteinase.In summary, our data support the hypothesis that as DOV13 cells interact with type I collagen, integrin receptors cluster on the cell surface, resulting in up-regulation of MT1-MMP and pro-MMP-2 processing, recruitment of MT1-MMP to sites of cell-matrix contact, MMP-2 surface association, and MT1-MMP-dependent collagen gel invasion. As a consequence of MT1-MMP collagenolysis, the resulting collagen cleavage products thermally denature, providing a substrate for a number of proteinases. In addition, MMP-2 is released from the cell surface to further advance matrix clearance through directed gelatinase activity on denatured collagen fragments. As α2β1 or α3β1integrin occupancy is reduced, collagen matrix stimulation of proteolysis is attenuated. Furthermore, MT1-MMP activity can be down-regulated by autolytic processing to a stable, inactive 43-kDa form that may functionally dilute productive enzyme-substrate interactions. Together, these data support an hypothesis wherein matrix status influences cell surface matrix-degrading potential to facilitate cellular functions including migration, invasion, and matrix remodeling. The MMP family is composed of at least 25 zinc-dependent extracellular endopeptidases whose activities are regulated predominantly by expression as inactive precursors, or zymogens (1Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2630) Google Scholar, 2Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar, 3Ellerbroek S.M. Stack M.S. BioEssays. 1999; 11: 940-949Crossref Scopus (133) Google Scholar). Although precise physiological activators of MMPs1 are unknown, a variety of serine proteinases and other MMPs in the extracellular milieu execute the initial propeptide cleavage eventsin vitro (2Nagase H. Biol. Chem. 1997; 378: 151-160PubMed Google Scholar). An exception to serine protease activation is pro-MMP-2 (72-kDa gelatinase A), which lacks the necessary basic amino acid cleavage sites in its pro-domain (4Corcoran M.L. Hewitt R.E. Kleiner Jr., D.E. Stetler-Stevenson W.G. Enzyme Protein. 1996; 49: 7-19Crossref PubMed Scopus (182) Google Scholar). A primary mechanism of pro-MMP-2 activation involves zymogen association with the cell surface via formation of a ternary complex containing tissue inhibitor of metalloproteinase (TIMP)-2 and membrane type 1-MMP (MT1-MMP, MMP-14) (3Ellerbroek S.M. Stack M.S. BioEssays. 1999; 11: 940-949Crossref Scopus (133) Google Scholar, 4Corcoran M.L. Hewitt R.E. Kleiner Jr., D.E. Stetler-Stevenson W.G. Enzyme Protein. 1996; 49: 7-19Crossref PubMed Scopus (182) Google Scholar, 5Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 6Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). Following trimeric complex formation, it is hypothesized that a neighboring MT1-MMP molecule that is not associated with TIMP-2 cleaves pro-MMP-2 at the Asn37–Leu38 peptide bond within the pro-domain (7Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Intermediately processed MMP-2 (Leu38–MMP-2) undergoes further concentration-dependent autolytic cleavage(s) to generate mature enzymes that can be released into the soluble phase or remain surface-associated (8Atkinson S.J. Crabbe T. Cowell S. Ward R.V. Butler M.J. Sato H. Seiki M. Reynolds J.J. Murphy G. J. Biol. Chem. 1995; 270: 30479-30485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Although biological mechanisms of active MMP-2 release from the cell surface are not well characterized and dissociation kinetics provide little insight, cellular binding affinities may shift following pro-MMP-2 cleavage. Culturing a variety of cell types within a three-dimensional gel of type I collagen stimulates cellular activation of pro-MMP-2 (9Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4544PubMed Google Scholar, 10Tomesek J.J. Halliday N.L. Updike D.L. Ahern-Moore J.S. Vu T.K. Liu R.W. Howard E.W. J. Biol. Chem. 1997; 272: 7482-7787Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Haas T.L. Davis S.J. Madri J.A. J. Biol. Chem. 1998; 273: 3604-3610Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar). Although MT1-MMP is implicated in MMP-2 processing, regulation of cellular events that promote MMP processing are poorly understood. As cellular interaction with type I collagen is mediated largely through integrin receptors, it has been postulated that collagen stimulation occurs either directly or indirectly through integrin signaling (12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar, 13Seltzer J.L. Lee A.Y. Akers K.T. Sudbeck B. Southon E.A. Wayner E.A. Eisen A.Z. Exp. Cell. Res. 1994; 213: 365-374Crossref PubMed Scopus (122) Google Scholar, 14Theret N. Lehti K. Musso O. Clement B. Hepatology. 1999; 30: 462-468Crossref PubMed Scopus (118) Google Scholar, 15Nguyen M. Arkell J. Jackson C.J. Int. J. Biochem. Cell Biol. 2000; 32: 621-631Crossref PubMed Scopus (41) Google Scholar, 16Boudreau N. Bissell M.J. Curr. Opin. Cell Biol. 1998; 10: 640-646Crossref PubMed Scopus (313) Google Scholar). In support, we have previously demonstrated that culturing DOV13 ovarian cancer cells in a three-dimensional collagen gel elicits a strong pro-MMP-2 activation response that can be mimicked by clustering of β1 integrin receptors (12Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar). Furthermore, pro-MMP-2 activation coincides with the processing of MT1-MMP into truncated 55- and 43-kDa forms on the cell surface. In this study, we utilize a variety of approaches to elucidate the biochemical requirements of type I collagen stimulation of MMP zymogen activation, characterize processed forms of MT1-MMP that are generated in this response, and examine the proteinase requirements for cellular invasion of type I collagen gels. Our findings illustrate a general mechanism by which cells may regulate cell surface-associated MMP activity via interactions with pericellular collagen matrix. DISCUSSIONMT1-MMP is a cell surface activator of pro-MMP-2 and has been implicated in collagen invasion and turnover (33Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 34Cockett M.I. Murphy G. Birch B.L. O'Connell J.P. Crabbe T. Millican A.T. Hart I.R. Docherty A.J. Biochem. Soc. Symp. 1998; 63: 295-313PubMed Google Scholar, 35Chambers A.F. Matrisian L.M. J. Natl. Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1426) Google Scholar, 36Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (507) Google Scholar). In this study, DOV13 ovarian cancer cells activate MT1-MMP as a consequence of culture in type I collagen gels and display MMP-dependent invasion of type I collagen, indicating that MMP activity is required for removal of collagen matrix constraints during invasion. However, migration over two-dimensional collagen is not impeded by MMP inhibitors. Although TIMP-1 does not interact with MT1-MMP, TIMP-2 specifically binds the proteinase, functioning in both inhibition and stabilization of the enzyme on the cell surface (26Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 32Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The ability of exogenous TIMP-2, in contrast to TIMP-1, to inhibit DOV13 collagen gel invasion implicates a cell surface proteolytic cascade initiated by MT1-MMP. This is further supported by data demonstrating that inhibition of MMP-2 cellular activation or collagen binding using rCD and rCBD (17
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