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• W2008796501 abstract "The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP·MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2. The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP·MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2. Recruitment of secreted proteases to the cell surface not only increases the proteolytic repertoire of a cell but also results in high local concentrations of the proteases. A level of control is achieved through focal proteolysis (1Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (420) Google Scholar), and this is of pivotal importance in many events mediated by matrix metalloproteinases (MMPs) 2The abbreviations used are: MMP, matrix metalloproteinase; MT-MMP, membrane type-MMP; sMT-MMP, soluble MT-MMP; TIMP, tissue inhibitor of matrix metalloproteinases; C domain, carboxyl domain; PBS, phosphatebuffered saline; DMEM, Dulbecco's modified essential medium; LCD, linker and hemopexin C domain; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MES, 4-morpholineethanesulfonic acid. such as tissue remodeling, angiogenesis, tumor metastasis, and leukocyte recruitment (2Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3903) Google Scholar, 3Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5169) Google Scholar). The MMP family is a large group of zinc-dependent endopeptidases comprising both secreted and membrane-anchored (referred to as membrane-type (MT)-MMPs) enzymes that are regulated by the tissue inhibitors of metalloproteinases (TIMPs). Control of secreted MMPs, such as MMP-2 (also known as gelatinase A), is also maintained through their expression as inactive zymogens, which require processing of the prodomain to attain full activity (4Gomis-Ruth F.X. Mol. Biotechnol. 2003; 24: 157-202Crossref PubMed Scopus (271) Google Scholar). Because of their location at the cell surface MT-MMPs, which include transmembrane-anchored MT-MMPs 1, 2, 3, and 5 (5Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2379) Google Scholar, 6Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 7Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 8Puente X. Pendas A. Llano E. Velasco G. Lopez-Otin C. Cancer Res. 1996; 56: 944-949PubMed Google Scholar) and glycosylphosphatidylinositol anchored MT-MMPs 4 and 6 (9Itoh Y. Kajita M. Kinoh H. Mori H. Okada A. Seiki M. J. Biol. Chem. 1999; 274: 34260-34266Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 10Kojima S. Itoh Y. Matsumoto S. Masuho Y. Seiki M. FEBS Lett. 2000; 480: 142-146Crossref PubMed Scopus (117) Google Scholar, 11Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar), play a major role in focal proteolysis. Despite the high degree of structural similarity of the MT-MMPs, differences in substrate specificity (12d'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 (387) Google Scholar, 13Shimada T. Nakamura H. Ohuchi E. Fujii Y. Murakami Y. Sato H. Seiki M. Okada Y. Eur. J. Biochem. 1999; 262: 907-914Crossref PubMed Scopus (88) Google Scholar, 14English W.R. Puente X.S. Freije J.M. Knauper V. Amour A. Merryweather A. Lopez-Otin C. Murphy G. J. Biol. Chem. 2000; 275: 14046-14055Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 15Wang Y. Johnson A.R. Ye Q.-Z. Dyer R.D. J. Biol. Chem. 1999; 274: 33043-33049Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), TIMP requirements for MMP-2 activation (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Zhao H. Bernardo M.M. Osenkowski P. Sohail A. Pei D. Nagase H. Kashiwagi M. Soloway P.D. DeClerck Y.A. Fridman R. J. Biol. Chem. 2004; 279: 8592-8601Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and tissue and cellular localization have been demonstrated (18Wong H. Muzik H. Groft L.L. Lafleur M.A. Matouk C. Forsyth P.A. Schultz G.A. Wall S.J. Edwards D.R. Methods Mol. Biol. 2001; 151: 305-320PubMed Google Scholar, 19Szabova L. Yamada S.S. Birkedal-Hansen H. Holmbeck K. J. Cell. Physiol. 2005; 205: 123-132Crossref PubMed Scopus (39) Google Scholar). The structural basis for these differences are not fully understood. The importance of MT1-MMP (MMP-14) and its role in MMP-2 activation and cell invasion have been extensively investigated (for reviews, see Refs. 20Holmbeck K. Bianco P. Yamada S. Birkedal-Hansen H. J. Cell. Physiol. 2004; 200: 11-19Crossref PubMed Scopus (150) Google Scholar, 21Sounni N.E. Noel A. Biochimie (Paris). 2005; 87: 329-342Crossref PubMed Scopus (124) Google Scholar, 22Sato H. Takino T. Miyamori H. Cancer Science. 2005; 96: 212-217Crossref PubMed Scopus (159) Google Scholar). In contrast, the role and function of MT2-MMP (MMP-15) is just beginning to be addressed. Studies using a cell line derived from a MT1-MMP knock-out mouse have shown the important contribution of MT2-MMP to cell invasion of fibrin matrices (23Hotary 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). Indeed, elevated expression of MT2-MMP has been reported in many cancers, such as glioblastomas (24Lampert K. Machein U. Machein M.R. Conca W. Peter H.H. Volk B. Am. J. Pathol. 1998; 153: 429-437Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 25Nakada M. Nakamura H. Ikeda E. Fujimoto N. Yamashita J. Sato H. Seiki M. Okada Y. Am. J. Pathol. 1999; 154: 417-428Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 26Zhang J. Sarkar S. Yong V.W. Carcinogenesis. 2005; 26: 2069-2077Crossref PubMed Scopus (76) Google Scholar), ovarian (27Davidson B. Goldberg I. Berner A. Nesland J.M. Givant-Horwitz V. Bryne M. Risberg B. Kristensen G.B. Trope C.G. Kopolovic J. Reich R. Am. J. Clin. Pathol. 2001; 115: 517-524Crossref PubMed Scopus (55) Google Scholar), urothelial (28Kitagawa Y. Kunimi K. Ito H. Sato H. Uchibayashi T. Okada Y. Seiki M. Namiki M. J. Urol. 1998; 160: 1540-1545Crossref PubMed Scopus (42) Google Scholar), and breast (19Szabova L. Yamada S.S. Birkedal-Hansen H. Holmbeck K. J. Cell. Physiol. 2005; 205: 123-132Crossref PubMed Scopus (39) Google Scholar, 29Ueno H. Nakamura H. Inoue M. Imai K. Noguchi M. Sato H. Seiki M. Okada Y. Cancer Res. 1997; 57: 2055-2060PubMed Google Scholar) carcinomas and correlated with increased invasiveness (25Nakada M. Nakamura H. Ikeda E. Fujimoto N. Yamashita J. Sato H. Seiki M. Okada Y. Am. J. Pathol. 1999; 154: 417-428Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 26Zhang J. Sarkar S. Yong V.W. Carcinogenesis. 2005; 26: 2069-2077Crossref PubMed Scopus (76) Google Scholar). In addition, MT2-MMP is involved in endothelial tubulogenesis (30Lafleur M.A. Handsley M.M. Knauper V. Murphy G. Edwards D.R. J. Cell Sci. 2002; 115: 3427-3438Crossref PubMed Google Scholar), malignant conversion of keratinocytes (31Mahloogi H. Bassi D.E. Klein-Szanto A.J. Carcinogenesis. 2002; 23: 565-572Crossref PubMed Google Scholar), and is an antiapoptotic factor (32Abraham R. Schafer J. Rothe M. Bange J. Knyazev P. Ullrich A. J. Biol. Chem. 2005; 280: 34123-34132Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). As well, MT2-MMP plays an important and distinct role from MT1-MMP in normal physiological processes (33Ogiwara K. Takano N. Shinohara M. Murakami M. Takahashi T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8442-8447Crossref PubMed Scopus (92) Google Scholar). MMP-2 is also a primary mediator of focal proteolysis due to its recruitment and activation at the cell surface. This occurs primarily through interaction of the MMP-2 hemopexin carboxyl (C) domain with the MT1-MMP/TIMP-2 receptor (34Zucker S. Drews M. Conner C. Foda H.D. DeClerck Y.A. Langley K.E. Bahou W.F. Docherty A.J.P. Cao J. J. Biol. Chem. 1998; 273: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 35Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. van Westrum S.S. Crabbe T. Clements J. d'Ortho M.-P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar) and the resultant formation of a trimolecular activation complex (5Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2379) Google Scholar, 36Strongin 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 (1438) Google Scholar, 37Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar). Activation of MMP-2 by MT1-MMP occurs in a two step process; the first cleavage within the MMP-2 prodomain (between Asn37-Leu38) is mediated by an active MT1-MMP molecule to generate the intermediate (68-kDa) form of MMP-2 (38Will 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 (505) Google Scholar, 39Sato H. Takino T. Kinoshita T. Imai K. Okada Y. Stevenson W.G.S. Seiki M. FEBS Lett. 1996; 385: 238-240Crossref PubMed Scopus (174) Google Scholar), and the second cleavage (between Asn80-Tyr81) is mediated in an autocatalytic manner in trans by active MMP-2 (38Will 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 (505) Google Scholar, 40Atkinson 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 (228) Google Scholar) and generates fully active MMP-2 (66-kDa). Because the second activation step was blocked by exogenous MMP-2 hemopexin C domain, it was concluded that it had to be mediated by a membrane-localized rather than soluble, active MMP-2 molecule (41Overall C.M. Tam E. McQuibban G.A. Morrison C. Wallon U.M. Bigg H.F. King A.E. Roberts C.R. J. Biol. Chem. 2000; 275: 39497-39506Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Whereas the first activation step of MMP-2 by MT1-MMP can occur in the absence of TIMP-2, the second activation step is absolutely TIMP-2-dependent(16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar,42Bigg H.F. Morrison C.J. Butler G.S. Bogoyevitch M.A. Wang Z. Soloway P.D. Overall C.M. Cancer Res. 2001; 61: 3610-3618PubMed Google Scholar). A number of alternative MT1-MMP-dependent activation pathways for MMP-2 have been reported involving proteases such as thrombin (43Lafleur M.A. Hollenberg M.D. Atkinson S.J. Knauper V. Murphy G. Edwards D.R. Biochem. J. 2001; 357: 107-115Crossref PubMed Scopus (113) Google Scholar), neutrophil elastase (44Shamamian P. Schwartz J.D. Pocock B.J. Monea S. Whiting D. Marcus S.G. Mignatti P. J. Cell. Physiol. 2001; 189: 197-206Crossref PubMed Scopus (295) Google Scholar), and plasmin (45Monea S. Lehti K. Keski-Oja J. Mignatti P. J. Cell. Physiol. 2002; 192: 160-170Crossref PubMed Scopus (134) Google Scholar), but the in vivo relevance of these pathways is unknown. Interestingly, a TIMP-independent MMP-2 activation pathway that is mediated by MT1-MMP and the tight-endothelial junction protein claudin-5 has been reported (46Miyamori H. Takino T. Kobayashi Y. Tokai H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: 28204-28211Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In addition, with the exception of MT4-MMP (14English W.R. Puente X.S. Freije J.M. Knauper V. Amour A. Merryweather A. Lopez-Otin C. Murphy G. J. Biol. Chem. 2000; 275: 14046-14055Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), all the other MT-MMPs have been shown to activate MMP-2 (7Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 47Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (512) Google Scholar, 48Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 49Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar). Using a TIMP-2-free cell line derived from the TIMP-2 knock-out mouse (50Wang Z. Juttermann R. Soloway P.D. J. Biol. Chem. 2000; 275: 26411-26415Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), we have shown that MT2-MMP activates MMP-2 in a TIMP-2-independent pathway that is clearly distinct from that of MT1-MMP (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Full activation of MMP-2 by MT2-MMP proceeded via the 68-kDa intermediate but occurred more rapidly than MT1-MMP-mediated activation. TIMP-2 had no enhancing effect on the level of active MMP-2 generated by MT2-MMP. In addition, activation by MT2-MMP required localization of MMP-2 at the cell surface in a MMP-2 hemopexin C domain-dependent but TIMP-2-independent manner. In this study we use Timp2-/- cells transfected for stable cell surface expression of MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP are exchanged to dissect the contribution that the different domains of MT2-MMP make to influence TIMP-independent activation of MMP-2. Recombinant Protein Expression and Purification Hemopexin C Domains—The cDNA encoding the linker (L) and hemopexin C domain (CD) (Thr305-Cys559) of MT2-MMP (MT2-MMP LCD) was amplified by PCR and cloned into the bacterial expression vector pGYMX (51Steffensen B. Wallon U.M. Overall C.M. J. Biol. Chem. 1995; 270: 11555-11566Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The pGYMX vectors containing MT2-MMP, MT1-MMP (41Overall C.M. Tam E. McQuibban G.A. Morrison C. Wallon U.M. Bigg H.F. King A.E. Roberts C.R. J. Biol. Chem. 2000; 275: 39497-39506Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and MMP-2 (52Wallon U.M. Overall C.M. J. Biol. Chem. 1997; 272: 7473-7481Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) LCD cDNAs were used to transform the Escherichia coli strain BL21 Gold, and cultures for protein production were grown in shaker flasks and a 100-liter fermenter (53Overall C.M. King A.E. Sam D.K. Ong A.D. Lau T.T. Wallon U.M. DeClerck Y.A. Atherstone J. J. Biol. Chem. 1999; 274: 4421-4429Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). His-tagged LCDs were extracted from inclusion bodies, re-folded, and purified using Ni2+-chelate chromatography (41Overall C.M. Tam E. McQuibban G.A. Morrison C. Wallon U.M. Bigg H.F. King A.E. Roberts C.R. J. Biol. Chem. 2000; 275: 39497-39506Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 53Overall C.M. King A.E. Sam D.K. Ong A.D. Lau T.T. Wallon U.M. DeClerck Y.A. Atherstone J. J. Biol. Chem. 1999; 274: 4421-4429Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Unlike MMP-2 and MT1-MMP LCDs, additional steps were required to purify MT2-MMP LCD to homogeneity. After elution of MT2-MMP LCD from the Ni2+-chelate column (GE Healthcare) (Vt = 40 ml) in 400 mm imidazole, the eluate was dialyzed into 20 mm phosphate buffer, pH 7.5, and loaded onto a CM-Sepharose column (GE Healthcare) (Vt = 5 ml). MT2-MMP LCD was eluted in 20 mm phosphate buffer, 1 m NaCl, pH 7.5. After dialysis into 20 mm 1,3-diaminopropane, pH 8.5, the MT2-MMP LCD was then loaded onto a Q-Sepharose column (GE Healthcare) (Vt = 2 ml) to remove contaminants, and the unbound fraction containing MT2-MMP LCD was collected. After increasing the pH to 10.9, it was reloaded onto a Q-Sepharose column (Vt = 2 ml), and the bound MT2-MMP LCD eluted using a 1 m NaCl gradient. Purified LCDs were stored in 10 mm Hepes, 150 mm NaCl, pH 7.2, and concentrations were determined using the predicted molar extinction coefficients. The identities of the LCDs were confirmed by Western blot analysis, N-terminal Edman sequencing, and MALDI-TOF mass spectrometry using a Voyager-DE STR Biospectrometry Work station (ABI) or electrospray ionization-TOF as previously described (54Tam E.M. Wu Y.I. Butler G.S. Stack M.S. Overall C.M. J. Biol. Chem. 2002; 277: 39005-39014Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Full-length MT-MMPs—Expression of full-length human MT1-MMP and MT2-MMP constructs in Timp2-/- skin fibroblasts was described in Morrison et al. (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The cDNA encoding full-length MT1-MMP containing a FLAG tag (DYK-DDDDK) inserted between Gly511-Gly512 in the stalk region of the protein (MT1-MMP FLAG) was kindly provided by Dr. Sharon Stack (Northwestern University, Chicago) (55Wu Y.I. Munshi H.G. Sen R. Snipas S.J. Salvesen G.S. Fridman R. Stack M.S. J. Biol. Chem. 2004; 279: 8278-8289Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Mutagenesis was used to introduce a FLAG sequence flanked by two Gly codons at 1753 bp in the MT2-MMP cDNA. This construct encodes MT2-MMP with the Gly-flanked FLAG tag inserted between Gly581-Gly582 residues in the stalk region of the protein (MT2-MMP FLAG). The double Gly residues at each end of the tag were designed for flexibility. The FLAG-tagged MT1-MMP and MT2-MMP constructs were used to generate chimera constructs, in which the 3′ end of MT1-MMP and MT2-MMP (after 957 and 1110 bp, respectively) were exchanged. This was achieved using PCR to introduce unique SspI sites (AATATT) by silent mutation in the MT1-MMP and MT2-MMP cDNAs. These sites were then used in cloning. The constructs generated encode proteins in which the C-terminal half of MT1-MMP and MT2-MMP is exchanged after Cys319 and Cys370, respectively. The resulting chimera proteins are termed MT1catL/MT2CD-MMP and MT2catL/MT1CD-MMP. All MT1-MMP and MT2-MMP constructs described were cloned into the pGW1GH vector and were used to transfect Timp2-/- fibroblasts using conditions described in Morrison et al. (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Positive clones were identified by flow cytometry using an anti-FLAG M2 antibody (Sigma) and by screening for MMP-2 activation in the presence and absence of 10 nm TIMP-2. Soluble MT-MMP (sMT-MMP)—PCR was used to introduce a FLAG sequence at the 3′ end of the sMT1-MMP cDNA used previously in Bigg et al. (42Bigg H.F. Morrison C.J. Butler G.S. Bogoyevitch M.A. Wang Z. Soloway P.D. Overall C.M. Cancer Res. 2001; 61: 3610-3618PubMed Google Scholar). This construct was cloned into the pPIC9 vector (Invitrogen) for expression in Pichia GS115 cells (Invitrogen). Cells were grown in 500-ml baffled shaker flask cultures, and after 24 h, 0.5% methanol was added to induce sMT1-MMP expression. Culture medium was diluted in MES buffer (final concentration, 50 mm MES, 5 mm CaCl2, 0.02% NaN3, 0.05% Brij, pH 6.2) and sMT1-MMP was purified upon binding to reactive red agarose (Sigma) (Vt = 30 ml). After washing, the column was eluted with 1 m NaCl in MES buffer. Fractions containing sMT1-MMP were pooled and dialyzed into 50 mm Hepes, 150 mm NaCl, pH 7.2. A cDNA construct was generated by PCR that encodes C-terminal FLAG-tagged sMT2-MMP truncated at Gln560. The construct was cloned into the pGW1GH vector and used to transfect Timp2-/- cells (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Positive clones with stable expression of sMT2-MMP were identified by Western blot analysis using an anti-FLAG M2 antibody (Sigma). Anti-FLAG M2 affinity gel (Sigma) was used to purify sMT2-MMP to homogeneity. TIMPs—Human TIMP-2 and TIMP-4 were expressed in Chinese hamster ovary cells and baby hamster kidney cells, respectively, and purified as described in Bigg et al. (42Bigg H.F. Morrison C.J. Butler G.S. Bogoyevitch M.A. Wang Z. Soloway P.D. Overall C.M. Cancer Res. 2001; 61: 3610-3618PubMed Google Scholar). Mouse TIMP-1 was expressed in Timp2-/- cells and purified as described in Shimokawa and Nagase (56Shimokawa K. Nagase H. Matrix Metalloproteinase Protocols. Humana Press Inc., Totowa, NJ2001: 275-304Google Scholar). TIMP-3 was kindly provided by Dr. Roy Black (Amgen, Seattle). All TIMPs were active site-titrated against a standard preparation of active MMP-8 to determine their concentrations. ProMMP-2—TIMP-2-free human proMMP-2 was expressed in Timp2-/- cells as previously described (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Cell Culture and MMP-2 Activation Assays—Timp2-/- cells were cultured, and cell assays were performed as described in Morrison et al. (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In brief, Timp2-/- cells expressing the full-length MT1-MMP and MT2-MMP constructs were seeded in 96-well plates at 2 × 104 cell/well. After 24 h of incubation at 37 °C, cells were washed extensively in PBS and incubated under serum-free conditions in the presence of 5 nm TIMP-2-free proMMP-2. In addition, the following were also added. (a) For hemopexin C domain competition assays, between 1 and 3750 nm MT2-MMP LCD, MT1-MMP LCD, and MMP-2 LCD pretreated with polymyxin B (10 μg/ml) (Sigma), (57Nisato R.E. Hosseini G. Sirrenberg C. Butler G.S. Crabbe T. Docherty A.J. Wiesner M. Murphy G. Overall C.M. Goodman S.L. Pepper M.S. Cancer Res. 2005; 65: 9377-9387Crossref PubMed Scopus (56) Google Scholar) to remove bacterial endotoxin contamination, were added to cells expressing MT2-MMP and MT1-MMP. TIMP-2 (10 nm) was added to cells expressing MT1-MMP. (b) For RGD peptide competition assays, between 0.05 and 12.5 μg/ml cyclic RGD or RAD peptide (kindly provided by Dr. Shoukat Dedhar, BC Cancer Agency, Vancouver, Canada) were added to cells expressing MT2-MMP. (c) Conditioned medium from Timp2-/- cells transfected with pGW1GH vector alone was concentrated using a Centricon centrifugal filtration device (Millepore) with a molecular weight cut-off of 10,000. The concentrated conditioned medium was then filter sterilized and added to cells expressing MT2-MMP and MT1-MMP. Cells expressing MT1-MMP were incubated with and without 10 nm TIMP-2. (d) TIMPs 1-4 were added to cells expressing MT2-MMP at concentrations between 0.01 and 270 nm, and TIMP-2 was added to cells expressing MT2catL/MT1CD-MMP, MT1catL/MT2CD-MMP, MT2-MMP FLAG, and MT1-MMP FLAG, at concentrations between 0.3 and 80 nm.(e) Neutralized Vitrogen® soluble type I collagen (Cohesion Technologies, Inc.) (between 12.5 and 200 μg/ml) was added to cells expressing MT2-MMP. Timp2-/- cells expressing MT2-MMP or transfected with pGW1GH vector alone were also grown on or in a native type I collagen matrix Vitrogen® (Cohesion Technologies, Inc.) (2.3 mg/ml). Neutralized collagen alone or collagen premixed with cells at 5 × 106 cells/ml (50 μl/well) were added to a 96-well plate, and the plate was incubated at 37 °C for 1 h to form collagen fibril gels. Then 100 μl/well of cells at 5 × 106 cells/ml in DMEM with 10% serum was added to wells containing collagen alone or to empty wells as a control. DMEM with 10% serum (100 μl/well) was added to the wells containing cells embedded in collagen. After 24 h of incubation at 37 °C, collagen gels with cells were washed extensively in PBS and incubated under serum-free conditions in the presence of 5 nm TIMP-2-free proMMP-2. In all cell assays, supernatants were collected after 24 h and analyzed by gelatin zymography using 8% polyacrylamide gels as described (16Morrison C.J. Butler G.S. Bigg H.F. Roberts C.R. Soloway P.D. Overall C.M. J. Biol. Chem. 2001; 276: 47402-47410Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Enzyme Capture Assay—Enzyme-linked immunosorbent assay plates were coated with 2 μg/ml sMT2-MMP, TIMP-2, or ovalbumin in Voller's buffer (15 mm Na2CO3,35mm NaHCO3, pH 9.6) for 16 h at 4 °C. Wells were then blocked for 2 h in PBS with 3% ovalbumin. After washing in PBS with 0.05% Tween, MMP-2 was added at concentrations between 15 and 2000 ng/well and incubated for 24 h at 4 °C. After extensive washing, 25 μl of non-reducing SDS-PAGE sample buffer was added per well to elute bound proteins. Samples were analyzed by gelatin zymography. Pulldown Assays—Gelatin-Sepharose was blocked in PBS with 0.5% bovine serum albumin for 2 h at 4°C. Then 10-μl aliquots of blocked gelatin-Sepharose were incubated with 100 μl of proMMP-2 (100 μg/ml) or with PBS for 2 h at 4 °C. After washing in PBS, 10-μl aliquots of gelatin-Sepharose with bound proMMP-2 were incubated with 100 μl of TIMP-2 (200 μg/ml) or concentrated conditioned medium or PBS or DMEM for 2 h at 4 °C. Then, after washing, the various samples were incubated with 100 μl of sMT1-MMP or sMT2-MMP (50 μg/ml) for 16 h at 4 °C. After extensive washing, gelatin-Sepharose beads were eluted with 30 μl of 10% Me2SO. Affi-Gel 10 (Bio-Rad) was coupled according to manufacturer's instructions to MMP-2 LCD or TIMP-2 as a positive control (1 mg protein/ml Affi-Gel) and then, after blocking reactive groups with 1 m ethanolamine, incubated with 100 μl of sMT1-MMP or sMT2-MMP (50 μg/ml) for 16 h at 4 °C. Bound proteins were removed from the Affi-Gel with denaturing SDS-PAGE sample buffer. Samples were analyzed by Western blotting using an anti-FLAG M2 antibody (Sigma)." @default.
• W2008796501 created "2016-06-24" @default.
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• W2008796501 date "2006-09-01" @default.
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• W2008796501 title "TIMP Independence of Matrix Metalloproteinase (MMP)-2 Activation by Membrane Type 2 (MT2)-MMP Is Determined by Contributions of Both the MT2-MMP Catalytic and Hemopexin C Domains" @default.
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• W2008796501 doi "https://doi.org/10.1074/jbc.m603331200" @default.
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