Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

TIMP-2 Is Required for Efficient Activation of proMMP-2 in Vivo

2000; Elsevier BV; Volume: 275; Issue: 34 Linguagem: Inglês

10.1074/jbc.m001270200

ISSN

1083-351X

Autores

Zhiping Wang, R Jüttermann, Paul D. Soloway,

Tópico(s)

Signaling Pathways in Disease

Resumo

Matrix metalloproteinases (MMPs) are synthesized as latent proenzymes. A proteolytic cleavage event involving processing of the cysteine-rich N-terminal propeptide is required for their full activation. Previous in vitro studies indicated that activation of proMMP-2 can occur through formation of a trimolecular complex between MMP-14, TIMP-2, and proMMP-2 at the cell surface. Using TIMP-2-deficient mice and cells derived from them, TIMP-2 was shown to be required for efficient proMMP-2 activation both in vivoand in vitro. The requirement for TIMP-2 was not cell-autonomous as exogenously added TIMP-2 could restore activation of proMMP-2 to TIMP-2-deficient cells. Mutant mice were overtly normal, viable, and fertile on the C57BL/6 background, indicating that both TIMP-2 and activated proMMP-2 are dispensable for normal development. Matrix metalloproteinases (MMPs) are synthesized as latent proenzymes. A proteolytic cleavage event involving processing of the cysteine-rich N-terminal propeptide is required for their full activation. Previous in vitro studies indicated that activation of proMMP-2 can occur through formation of a trimolecular complex between MMP-14, TIMP-2, and proMMP-2 at the cell surface. Using TIMP-2-deficient mice and cells derived from them, TIMP-2 was shown to be required for efficient proMMP-2 activation both in vivoand in vitro. The requirement for TIMP-2 was not cell-autonomous as exogenously added TIMP-2 could restore activation of proMMP-2 to TIMP-2-deficient cells. Mutant mice were overtly normal, viable, and fertile on the C57BL/6 background, indicating that both TIMP-2 and activated proMMP-2 are dispensable for normal development. matrix metalloproteinase tissue inhibitors of metalloproteinase concanavalin A 4-aminophenyl mercuric acetate More than 20 matrix metalloproteinases (MMPs)1 have been described that collectively degrade all extracellular matrix and a number of non-matrix proteins involved in inflammation and cell growth control (reviewed in Ref. 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). The MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) have been associated directly and indirectly with many developmental processes such as branching morphogenesis (2Sympson C.J. Talhouk R.S. Alexander C.M. Chin J.R. Clift S.M. Bissell M.J. Werb Z. J. Cell Biol. 1994; 125: 681-693Crossref PubMed Scopus (348) Google Scholar, 3Lelongt B. Trugnan G. Murphy G. Ronco P.M. J. Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (148) Google Scholar, 4Koochekpour S. Jeffers M. Wang P.H. Gong C. Taylor G.A. Roessler L.M. Stearman R. Vasselli J.R. Stetler-Stevenson W.G. Kaelin Jr., W.G. Linehan W.M. Klausner R.D. Gnarra J.R. Vande Woude G.F. Mol. Cell. Biol. 1999; 19 (12): 5902Crossref PubMed Scopus (179) Google Scholar, 5Fata J.E. Leco K.J. Moorehead R.A. Martin D.C. Khokha R. Dev. Biol. 1999; 211: 238-254Crossref PubMed Scopus (91) Google Scholar, 6Barasch J. Yang J. Qiao J. Tempst P. Erdjument-Bromage H. Leung W. Oliver J.A. J. Clin. Invest. 1999; 103: 1299-1307Crossref PubMed Scopus (94) Google Scholar), regulation of cell migration (7Mignatti P. Robbins E. Rifkin D.B. Cell. 1986; 47: 487-498Abstract Full Text PDF PubMed Scopus (636) Google Scholar), apoptosis (8Guedez L. Stetler-Stevenson W.G. Wolff L. Wang J. Fukushima P. Mansoor A. Stetler-Stevenson M. J. Clin. Invest. 1998; 102: 2002-2010Crossref PubMed Scopus (361) Google Scholar, 9Boudreau N. Sympson C.J. Werb Z. Bissell M.J. Science. 1995; 267: 891-893Crossref PubMed Scopus (1113) Google Scholar), angiogenesis (10Moses M.A. Langer R. J. Cell. Biol. 1991; 47: 230-235Google Scholar), and regulation of innate immunity (11Osiewicz K. McGarry M. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878: 494-496Crossref PubMed Scopus (13) Google Scholar). 2B.-J. Yoon, K. Osiewicz, B. Johnston, K. Weaver, W. Potter, M. J. Preston, R. Jaenisch, G. B. Pier, T. Dougherty, P. Kubes, and P. D. Soloway, submitted for publication. 2B.-J. Yoon, K. Osiewicz, B. Johnston, K. Weaver, W. Potter, M. J. Preston, R. Jaenisch, G. B. Pier, T. Dougherty, P. Kubes, and P. D. Soloway, submitted for publication. In addition to their link with developmental events, MMPs have been implicated in several disease processes such as tumor metastasis (12Liotta L.A. Tryggvason K. Garbisa S. Hart I. Foltz C.M. Shafie S. Nature. 1980; 284: 67-68Crossref PubMed Scopus (1549) Google Scholar), arthritis (13Jubb R.W. Fell H.B. J. Pathol. 1980; 130: 159-167Crossref PubMed Scopus (86) Google Scholar), and emphysema (14D'Armiento J. Dalal S.S. Okada Y. Berg R.A. Chada K. Cell. 1992; 71: 955-961Abstract Full Text PDF PubMed Scopus (325) Google Scholar, 15Hautamaki R.D. Kobayashi D.K. Senior R.M. Shapiro S.D. Science. 1997; 277: 2002-2004Crossref PubMed Scopus (1233) Google Scholar). They are regulated in many ways that include transcriptional (16Overall C.M. Wrana J.L. Sodek J. J. Biol. Chem. 1991; 266: 14064-14071Abstract Full Text PDF PubMed Google Scholar) and post-translational mechanisms. Two post-translational means of MMP regulation have been described. First, MMPs are synthesized as latent pro-enzymes. A cysteine-rich N-terminal peptide of the latent proenzyme interacts with the Zn+2 at the active site, blocking proteolytic activity of the proteinase (17Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1192) Google Scholar). After cleavage of the propeptide, the latent enzyme becomes activated, enabling it to degrade its substrate. A second mode of post-translational regulation has been described as well. Active MMPs are inhibited by the broad spectrum proteinase inhibitor α2-macroglobulin (18Abe S. Nagai Y. J. Biochem. 1973; 73: 897-900Crossref PubMed Scopus (66) Google Scholar) and by four known MMP-specific tissue inhibitors of metalloproteinases or TIMPs (19Murphy G. McGuire M.B. Russell R.G. Reynolds J.J. Clin. Sci. 1981; 61: 711-716Crossref PubMed Scopus (36) Google Scholar, 20Stetler-Stevenson W.G. Krutzsch H.C. Liotta L.A. J. Biol. Chem. 1989; 264: 17374-17378Abstract Full Text PDF PubMed Google Scholar, 21Pavloff N. Staskus P.W. Kishnani N.S. Hawkes S.P. J. Biol. Chem. 1992; 267: 17321-17326Abstract Full Text PDF PubMed Google Scholar, 22Greene J. Wang M. Liu Y.E. Raymond L.A. Rosen C. Shi Y.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). The mechanisms by which MMPs are proteolytically processed from their latent to their activated forms and how they are regulated by inhibitors are important to understanding their contributions to normal developmental and pathological processes.Evidence exists for several mechanisms of proteolytic activation of the latent proMMPs. For example, plasmin can participate in the activation of proMMP-1(23Okada Y. Morodomi T. Enghild J.J. Suzuki K. Yasui A. Nakanishi I. Salvesen G. Nagase H. Eur. J. Biochem. 1990; 194: 721-730Crossref PubMed Scopus (383) Google Scholar), -3 (24Nagase H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Crossref PubMed Scopus (350) Google Scholar), -7 (25Imai K. Yokohama Y. Nakanishi I. Ohuchi E. Fujii Y. Nakai N. Okada Y. J. Biol. Chem. 1995; 270: 6691-6697Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), -9 (26Ramos-DeSimone N. Hahn-Dantona E. Sipley J. Nagase H. French D.L. Quigley J.P. J. Biol. Chem. 1999; 274: 13066-13076Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 27Baramova E.N. Bajou K. Remacle A. L'Hoir C. Krell H.W. Weidle U.H. Noel A. Foidart J.M. FEBS Lett. 1997; 405 (62): 157Crossref PubMed Scopus (240) Google Scholar), -13 (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar), and -14 (29Okumura Y. Sato H. Seiki M. Kido H. FEBS Lett. 1997; 402: 181-184Crossref PubMed Scopus (113) Google Scholar), whereas furin can activate the membrane-associated proMMP-14 (30Sato H. Kinoshita T. Takino T. Nakayama K. Seiki M. FEBS Lett. 1996; 393: 101-104Crossref PubMed Scopus (298) Google Scholar), proMMP-11 (31Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (530) Google Scholar), and others. Already activated MMPs can also contribute to activation of other proMMPs. For example, MMP-3 can activate proMMP-1 (32Suzuki K. Enghild J.J. Morodomi T. Salvesen G. Nagase H. Biochemistry. 1990; 29: 10261-10270Crossref PubMed Scopus (384) Google Scholar), and -proMMP-7 (25Imai K. Yokohama Y. Nakanishi I. Ohuchi E. Fujii Y. Nakai N. Okada Y. J. Biol. Chem. 1995; 270: 6691-6697Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) and MMP-14 together with MMP-2 can contribute to the activation of proMMP-13 by a cascade mechanism (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar).In biochemical studies designed to elucidate the mechanisms of proMMP-2 activation, it was shown that membrane fractions from proMMP-2-activating cells were able to catalyze the proteolytic processing of latent proMMP-2 and that both the membrane-associated MMP-14 and TIMP-2 participate in the activation (33Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 34Atkinson 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, 35Strongin 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). This process of activation first involves an initial cleavage of proMMP-2 by MMP-14 at the Asn37-Leu38 bond of proMMP-2 followed by an autocatalytic cleavage of the intermediate product at the Asn80-Tyr81 bond, generating the fully active MMP-2 (36Will 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). Although TIMP-2 is an inhibitor of MMPs, paradoxically in this mechanism it can function as a co-activator of proMMP-2 as well. Evidence indicates that TIMP-2 participates in the activation of proMMP-2 by MMP-14 by facilitating the assembly of a complex on the cell surface that brings the two MMPs together. In this trimeric complex, the C termini of proMMP-2 and TIMP-2 interact while the N termini of MMP-14 and TIMP-2 interact (34Atkinson 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, 35Strongin 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, 36Will 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, 37Butler 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 (538) Google Scholar, 38Butler G.S. Will H. Atkinson S.J. Murphy G. Eur. J. Biochem. 1997; 244: 653-657Crossref PubMed Scopus (145) Google Scholar, 39Cao J. Sato H. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 40Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (480) Google Scholar, 41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). The ability of TIMP-2 to participate in this mechanism is sensitive to the local extracellular matrix environment (42Stanton H. Gavrilovic J. Atkinson S.J. d'Ortho M.P. Yamada K.M. Zardi L. Murphy G. J. Cell Sci. 1998; 111: 2789-2798Crossref PubMed Google Scholar, 43Itoh Y. Binner S. Nagase H. Biochem. J. 1995; 308: 645-651Crossref PubMed Scopus (99) Google Scholar). Furthermore, TIMP-2 may be completely dispensable for proMMP-2 activation since the extracellular domain of MMP-14 can lead to proMMP-2 activation in the absence of any TIMP-2 (44Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Such TIMP-2-independent pathways may exist that allow intracellular activation of latent proMMP-2 (45Lee A.Y. Akers K.T. Collier M. Li L. Eisen A.Z. Seltzer J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4424-4429Crossref PubMed Scopus (59) Google Scholar).If the TIMP-2-dependent mechanism of proMMP-2 activation is the primary means for activating the latent proenzyme in normal tissues or cultured primary cells, then conversion of the latent MMP-2 proenzyme to its active form would fail in tissues or cells deficient for TIMP-2. To test this hypothesis and to study other in vivo functions of TIMP-2 in normal development and disease processes, mice carrying a null mutation in the single gene encodingTimp2 on chromosome 11 were generated. Reported here is the development of TIMP-2-deficient mice and studies of proMMP-2 activation in tissues and cells derived from mutant animals.DISCUSSIONMice carrying a targeted mutation in Timp2 were generated. No Timp2 mRNA was detected in tissues from mutant animals, indicating the mutation was null. Loss of TIMP-2 did not adversely affect normal mouse development, viability, or fertility on the C57BL/6 background. However, loss of the inhibitor did result in a dramatic reduction in activation of the latent proenzyme, proMMP-2, in lung tissue in vivo and cultured primary fibroblastsin vitro. This demonstrates that TIMP-2 is not only a potential component of the proMMP-2 activation mechanism, as indicated by several published reports, but in fact it is required for normal activation of the latent proenzyme in vivo. It cannot be ruled out that TIMP-2-independent mechanisms exist for activating proMMP-2. However, if such mechanisms exist, they are far less efficient at inducing proMMP-2 activation than the mechanism involving TIMP-2 in the tissue and cells analyzed in this study. Failure of proMMP-2 activation in and of itself was expected to have minimal developmental consequences, consistent with the lack of a developmental phenotype in mice deficient for MMP-2 (50Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res. 1998; 58: 1048-1051PubMed Google Scholar). However, any phenotypes seen in MMP-2-deficient mice may also be expected to be seen in TIMP-2-deficient mice.Because the proMMP-2 activation defect observed in cultures ofTimp2 mutant cells could be suppressed by exogenously added recombinant human TIMP-2 protein, the requirement for TIMP-2 in proMMP-2 activation is not cell autonomous. The effects of added TIMP-2 protein on proMMP-2 activation in the culture systems used were either stimulatory or inhibitory, depending upon the concentration of TIMP-2 protein in the cultures. This is consistent with observations that the stoichiometry of the various co-activators and of the latent proMMP-2 protein is critical in governing proMMP-2 activation. Although exogenously added human TIMP-2 could partly restore proMMP-2 activation to homozygous mutant cells, the activation products that accumulated were intermediates and not fully active MMP-2. The reasons for the partial activation are not clear. There may be important species differences between mouse and human TIMP-2. The specific preparations of human TIMP-2 used may have been only partly active. Finally, the cellular location where TIMP-2 interacts with proMMP-2 may be important to the nature of the interaction and the capacity of proMMP-2 to subsequently undergo complete activation. Exogenously added TIMP-2 may not provide the same interaction with proMMP-2 as endogenously produced protein.TIMP-1 was unable to replace TIMP-2 as a co-factor for proMMP-2 activation; however, it was able to block proMMP-2 activation at higher concentrations. This indicated that MMP activity is itself needed for proMMP-2 activation and also highlights the functional differences between TIMPs 1 and 2. These differences are likely to result from the particular structural features of the two TIMPs. One specific feature that may allow TIMP-2, but not TIMP-1, to participate in proMMP-2 activation is the 10-residue-long negatively charged C-terminal tail of TIMP-2, which is only 3 residues long in TIMP-1 (41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). This tail may enable TIMP-2 to form salt bridges with specific positively charged residues at the junction of C-terminal hemopexin domains III and IV of pro-MMP-2, which are required for proMMP-2 to form a complex with TIMP-2 (37Butler 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 (538) Google Scholar, 51Overall 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). A second structural difference between TIMPs 1 and 2 that may enable TIMP-2 but not TIMP-1 to participate in proMMP-2 activation lies at the N terminus. Residues within the unique long N-terminal A-B β-hairpin loop of TIMP-2 are required for specific interactions with MMP-14 (52Butler G.S. Hutton M. Wattam B.A. Williamson R.A. Knauper V. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 20391-20396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). TIMP-1 is 7 residues shorter in this domain, which significantly shortens the A-B β-hairpin. This and other loop structure differences between the two TIMPs cause them to associate very differently with MMPs and may prevent TIMP-1 from interacting with MMP-14 in a way that enables it to replace TIMP-2 as a co-activator of proMMP-2 (41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar).Activation mechanisms for proMMPs other than proMMP-2 have been described as cell surface events requiring membrane type MMPs. For example, like proMMP-2, proMMP-13 can be activated by a mechanism involving MMP-14 and MMP-2 (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). It is not yet known if efficient proMMP-13 activation, like proMMP-2 activation, is dependent upon the presence of TIMP-2. These studies are ongoing. Other TIMPs may also play a direct role in activation of proMMPs. TIMP-4 is able to interact with the C-terminal region of proMMP-2 in a manner similar to that of TIMP-2 (53Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar). Whether it, like TIMP-2, is required for efficient proMMP-2 activation is not known.Other important functional differences between TIMPs 1 and 2 have been revealed during analysis of mutant mice. Although TIMP-2 loss resulted in defects in proMMP-2 activation, this was unaffected by a mutation within Timp1. Also, although TIMP-1-deficient animals are hyper-resistant to corneal infections with Pseudomonas aeruginosa by a complement-dependent mechanism, TIMP-2-deficient mice have normal immune responses (11Osiewicz K. McGarry M. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878: 494-496Crossref PubMed Scopus (13) Google Scholar, 54Wang Z. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878 (21): 519Crossref PubMed Scopus (7) Google Scholar). It is possible, however, that other MMP-sensitive immune response mechanisms, possibly involving defensin production (55Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (903) Google Scholar), are altered. More than 20 matrix metalloproteinases (MMPs)1 have been described that collectively degrade all extracellular matrix and a number of non-matrix proteins involved in inflammation and cell growth control (reviewed in Ref. 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). The MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) have been associated directly and indirectly with many developmental processes such as branching morphogenesis (2Sympson C.J. Talhouk R.S. Alexander C.M. Chin J.R. Clift S.M. Bissell M.J. Werb Z. J. Cell Biol. 1994; 125: 681-693Crossref PubMed Scopus (348) Google Scholar, 3Lelongt B. Trugnan G. Murphy G. Ronco P.M. J. Cell Biol. 1997; 136: 1363-1373Crossref PubMed Scopus (148) Google Scholar, 4Koochekpour S. Jeffers M. Wang P.H. Gong C. Taylor G.A. Roessler L.M. Stearman R. Vasselli J.R. Stetler-Stevenson W.G. Kaelin Jr., W.G. Linehan W.M. Klausner R.D. Gnarra J.R. Vande Woude G.F. Mol. Cell. Biol. 1999; 19 (12): 5902Crossref PubMed Scopus (179) Google Scholar, 5Fata J.E. Leco K.J. Moorehead R.A. Martin D.C. Khokha R. Dev. Biol. 1999; 211: 238-254Crossref PubMed Scopus (91) Google Scholar, 6Barasch J. Yang J. Qiao J. Tempst P. Erdjument-Bromage H. Leung W. Oliver J.A. J. Clin. Invest. 1999; 103: 1299-1307Crossref PubMed Scopus (94) Google Scholar), regulation of cell migration (7Mignatti P. Robbins E. Rifkin D.B. Cell. 1986; 47: 487-498Abstract Full Text PDF PubMed Scopus (636) Google Scholar), apoptosis (8Guedez L. Stetler-Stevenson W.G. Wolff L. Wang J. Fukushima P. Mansoor A. Stetler-Stevenson M. J. Clin. Invest. 1998; 102: 2002-2010Crossref PubMed Scopus (361) Google Scholar, 9Boudreau N. Sympson C.J. Werb Z. Bissell M.J. Science. 1995; 267: 891-893Crossref PubMed Scopus (1113) Google Scholar), angiogenesis (10Moses M.A. Langer R. J. Cell. Biol. 1991; 47: 230-235Google Scholar), and regulation of innate immunity (11Osiewicz K. McGarry M. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878: 494-496Crossref PubMed Scopus (13) Google Scholar). 2B.-J. Yoon, K. Osiewicz, B. Johnston, K. Weaver, W. Potter, M. J. Preston, R. Jaenisch, G. B. Pier, T. Dougherty, P. Kubes, and P. D. Soloway, submitted for publication. 2B.-J. Yoon, K. Osiewicz, B. Johnston, K. Weaver, W. Potter, M. J. Preston, R. Jaenisch, G. B. Pier, T. Dougherty, P. Kubes, and P. D. Soloway, submitted for publication. In addition to their link with developmental events, MMPs have been implicated in several disease processes such as tumor metastasis (12Liotta L.A. Tryggvason K. Garbisa S. Hart I. Foltz C.M. Shafie S. Nature. 1980; 284: 67-68Crossref PubMed Scopus (1549) Google Scholar), arthritis (13Jubb R.W. Fell H.B. J. Pathol. 1980; 130: 159-167Crossref PubMed Scopus (86) Google Scholar), and emphysema (14D'Armiento J. Dalal S.S. Okada Y. Berg R.A. Chada K. Cell. 1992; 71: 955-961Abstract Full Text PDF PubMed Scopus (325) Google Scholar, 15Hautamaki R.D. Kobayashi D.K. Senior R.M. Shapiro S.D. Science. 1997; 277: 2002-2004Crossref PubMed Scopus (1233) Google Scholar). They are regulated in many ways that include transcriptional (16Overall C.M. Wrana J.L. Sodek J. J. Biol. Chem. 1991; 266: 14064-14071Abstract Full Text PDF PubMed Google Scholar) and post-translational mechanisms. Two post-translational means of MMP regulation have been described. First, MMPs are synthesized as latent pro-enzymes. A cysteine-rich N-terminal peptide of the latent proenzyme interacts with the Zn+2 at the active site, blocking proteolytic activity of the proteinase (17Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1192) Google Scholar). After cleavage of the propeptide, the latent enzyme becomes activated, enabling it to degrade its substrate. A second mode of post-translational regulation has been described as well. Active MMPs are inhibited by the broad spectrum proteinase inhibitor α2-macroglobulin (18Abe S. Nagai Y. J. Biochem. 1973; 73: 897-900Crossref PubMed Scopus (66) Google Scholar) and by four known MMP-specific tissue inhibitors of metalloproteinases or TIMPs (19Murphy G. McGuire M.B. Russell R.G. Reynolds J.J. Clin. Sci. 1981; 61: 711-716Crossref PubMed Scopus (36) Google Scholar, 20Stetler-Stevenson W.G. Krutzsch H.C. Liotta L.A. J. Biol. Chem. 1989; 264: 17374-17378Abstract Full Text PDF PubMed Google Scholar, 21Pavloff N. Staskus P.W. Kishnani N.S. Hawkes S.P. J. Biol. Chem. 1992; 267: 17321-17326Abstract Full Text PDF PubMed Google Scholar, 22Greene J. Wang M. Liu Y.E. Raymond L.A. Rosen C. Shi Y.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). The mechanisms by which MMPs are proteolytically processed from their latent to their activated forms and how they are regulated by inhibitors are important to understanding their contributions to normal developmental and pathological processes. Evidence exists for several mechanisms of proteolytic activation of the latent proMMPs. For example, plasmin can participate in the activation of proMMP-1(23Okada Y. Morodomi T. Enghild J.J. Suzuki K. Yasui A. Nakanishi I. Salvesen G. Nagase H. Eur. J. Biochem. 1990; 194: 721-730Crossref PubMed Scopus (383) Google Scholar), -3 (24Nagase H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Crossref PubMed Scopus (350) Google Scholar), -7 (25Imai K. Yokohama Y. Nakanishi I. Ohuchi E. Fujii Y. Nakai N. Okada Y. J. Biol. Chem. 1995; 270: 6691-6697Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), -9 (26Ramos-DeSimone N. Hahn-Dantona E. Sipley J. Nagase H. French D.L. Quigley J.P. J. Biol. Chem. 1999; 274: 13066-13076Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 27Baramova E.N. Bajou K. Remacle A. L'Hoir C. Krell H.W. Weidle U.H. Noel A. Foidart J.M. FEBS Lett. 1997; 405 (62): 157Crossref PubMed Scopus (240) Google Scholar), -13 (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar), and -14 (29Okumura Y. Sato H. Seiki M. Kido H. FEBS Lett. 1997; 402: 181-184Crossref PubMed Scopus (113) Google Scholar), whereas furin can activate the membrane-associated proMMP-14 (30Sato H. Kinoshita T. Takino T. Nakayama K. Seiki M. FEBS Lett. 1996; 393: 101-104Crossref PubMed Scopus (298) Google Scholar), proMMP-11 (31Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (530) Google Scholar), and others. Already activated MMPs can also contribute to activation of other proMMPs. For example, MMP-3 can activate proMMP-1 (32Suzuki K. Enghild J.J. Morodomi T. Salvesen G. Nagase H. Biochemistry. 1990; 29: 10261-10270Crossref PubMed Scopus (384) Google Scholar), and -proMMP-7 (25Imai K. Yokohama Y. Nakanishi I. Ohuchi E. Fujii Y. Nakai N. Okada Y. J. Biol. Chem. 1995; 270: 6691-6697Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) and MMP-14 together with MMP-2 can contribute to the activation of proMMP-13 by a cascade mechanism (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). In biochemical studies designed to elucidate the mechanisms of proMMP-2 activation, it was shown that membrane fractions from proMMP-2-activating cells were able to catalyze the proteolytic processing of latent proMMP-2 and that both the membrane-associated MMP-14 and TIMP-2 participate in the activation (33Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 34Atkinson 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, 35Strongin 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). This process of activation first involves an initial cleavage of proMMP-2 by MMP-14 at the Asn37-Leu38 bond of proMMP-2 followed by an autocatalytic cleavage of the intermediate product at the Asn80-Tyr81 bond, generating the fully active MMP-2 (36Will 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). Although TIMP-2 is an inhibitor of MMPs, paradoxically in this mechanism it can function as a co-activator of proMMP-2 as well. Evidence indicates that TIMP-2 participates in the activation of proMMP-2 by MMP-14 by facilitating the assembly of a complex on the cell surface that brings the two MMPs together. In this trimeric complex, the C termini of proMMP-2 and TIMP-2 interact while the N termini of MMP-14 and TIMP-2 interact (34Atkinson 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, 35Strongin 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, 36Will 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, 37Butler 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 (538) Google Scholar, 38Butler G.S. Will H. Atkinson S.J. Murphy G. Eur. J. Biochem. 1997; 244: 653-657Crossref PubMed Scopus (145) Google Scholar, 39Cao J. Sato H. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 40Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (480) Google Scholar, 41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). The ability of TIMP-2 to participate in this mechanism is sensitive to the local extracellular matrix environment (42Stanton H. Gavrilovic J. Atkinson S.J. d'Ortho M.P. Yamada K.M. Zardi L. Murphy G. J. Cell Sci. 1998; 111: 2789-2798Crossref PubMed Google Scholar, 43Itoh Y. Binner S. Nagase H. Biochem. J. 1995; 308: 645-651Crossref PubMed Scopus (99) Google Scholar). Furthermore, TIMP-2 may be completely dispensable for proMMP-2 activation since the extracellular domain of MMP-14 can lead to proMMP-2 activation in the absence of any TIMP-2 (44Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Such TIMP-2-independent pathways may exist that allow intracellular activation of latent proMMP-2 (45Lee A.Y. Akers K.T. Collier M. Li L. Eisen A.Z. Seltzer J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4424-4429Crossref PubMed Scopus (59) Google Scholar). If the TIMP-2-dependent mechanism of proMMP-2 activation is the primary means for activating the latent proenzyme in normal tissues or cultured primary cells, then conversion of the latent MMP-2 proenzyme to its active form would fail in tissues or cells deficient for TIMP-2. To test this hypothesis and to study other in vivo functions of TIMP-2 in normal development and disease processes, mice carrying a null mutation in the single gene encodingTimp2 on chromosome 11 were generated. Reported here is the development of TIMP-2-deficient mice and studies of proMMP-2 activation in tissues and cells derived from mutant animals. DISCUSSIONMice carrying a targeted mutation in Timp2 were generated. No Timp2 mRNA was detected in tissues from mutant animals, indicating the mutation was null. Loss of TIMP-2 did not adversely affect normal mouse development, viability, or fertility on the C57BL/6 background. However, loss of the inhibitor did result in a dramatic reduction in activation of the latent proenzyme, proMMP-2, in lung tissue in vivo and cultured primary fibroblastsin vitro. This demonstrates that TIMP-2 is not only a potential component of the proMMP-2 activation mechanism, as indicated by several published reports, but in fact it is required for normal activation of the latent proenzyme in vivo. It cannot be ruled out that TIMP-2-independent mechanisms exist for activating proMMP-2. However, if such mechanisms exist, they are far less efficient at inducing proMMP-2 activation than the mechanism involving TIMP-2 in the tissue and cells analyzed in this study. Failure of proMMP-2 activation in and of itself was expected to have minimal developmental consequences, consistent with the lack of a developmental phenotype in mice deficient for MMP-2 (50Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res. 1998; 58: 1048-1051PubMed Google Scholar). However, any phenotypes seen in MMP-2-deficient mice may also be expected to be seen in TIMP-2-deficient mice.Because the proMMP-2 activation defect observed in cultures ofTimp2 mutant cells could be suppressed by exogenously added recombinant human TIMP-2 protein, the requirement for TIMP-2 in proMMP-2 activation is not cell autonomous. The effects of added TIMP-2 protein on proMMP-2 activation in the culture systems used were either stimulatory or inhibitory, depending upon the concentration of TIMP-2 protein in the cultures. This is consistent with observations that the stoichiometry of the various co-activators and of the latent proMMP-2 protein is critical in governing proMMP-2 activation. Although exogenously added human TIMP-2 could partly restore proMMP-2 activation to homozygous mutant cells, the activation products that accumulated were intermediates and not fully active MMP-2. The reasons for the partial activation are not clear. There may be important species differences between mouse and human TIMP-2. The specific preparations of human TIMP-2 used may have been only partly active. Finally, the cellular location where TIMP-2 interacts with proMMP-2 may be important to the nature of the interaction and the capacity of proMMP-2 to subsequently undergo complete activation. Exogenously added TIMP-2 may not provide the same interaction with proMMP-2 as endogenously produced protein.TIMP-1 was unable to replace TIMP-2 as a co-factor for proMMP-2 activation; however, it was able to block proMMP-2 activation at higher concentrations. This indicated that MMP activity is itself needed for proMMP-2 activation and also highlights the functional differences between TIMPs 1 and 2. These differences are likely to result from the particular structural features of the two TIMPs. One specific feature that may allow TIMP-2, but not TIMP-1, to participate in proMMP-2 activation is the 10-residue-long negatively charged C-terminal tail of TIMP-2, which is only 3 residues long in TIMP-1 (41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). This tail may enable TIMP-2 to form salt bridges with specific positively charged residues at the junction of C-terminal hemopexin domains III and IV of pro-MMP-2, which are required for proMMP-2 to form a complex with TIMP-2 (37Butler 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 (538) Google Scholar, 51Overall 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). A second structural difference between TIMPs 1 and 2 that may enable TIMP-2 but not TIMP-1 to participate in proMMP-2 activation lies at the N terminus. Residues within the unique long N-terminal A-B β-hairpin loop of TIMP-2 are required for specific interactions with MMP-14 (52Butler G.S. Hutton M. Wattam B.A. Williamson R.A. Knauper V. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 20391-20396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). TIMP-1 is 7 residues shorter in this domain, which significantly shortens the A-B β-hairpin. This and other loop structure differences between the two TIMPs cause them to associate very differently with MMPs and may prevent TIMP-1 from interacting with MMP-14 in a way that enables it to replace TIMP-2 as a co-activator of proMMP-2 (41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar).Activation mechanisms for proMMPs other than proMMP-2 have been described as cell surface events requiring membrane type MMPs. For example, like proMMP-2, proMMP-13 can be activated by a mechanism involving MMP-14 and MMP-2 (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). It is not yet known if efficient proMMP-13 activation, like proMMP-2 activation, is dependent upon the presence of TIMP-2. These studies are ongoing. Other TIMPs may also play a direct role in activation of proMMPs. TIMP-4 is able to interact with the C-terminal region of proMMP-2 in a manner similar to that of TIMP-2 (53Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar). Whether it, like TIMP-2, is required for efficient proMMP-2 activation is not known.Other important functional differences between TIMPs 1 and 2 have been revealed during analysis of mutant mice. Although TIMP-2 loss resulted in defects in proMMP-2 activation, this was unaffected by a mutation within Timp1. Also, although TIMP-1-deficient animals are hyper-resistant to corneal infections with Pseudomonas aeruginosa by a complement-dependent mechanism, TIMP-2-deficient mice have normal immune responses (11Osiewicz K. McGarry M. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878: 494-496Crossref PubMed Scopus (13) Google Scholar, 54Wang Z. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878 (21): 519Crossref PubMed Scopus (7) Google Scholar). It is possible, however, that other MMP-sensitive immune response mechanisms, possibly involving defensin production (55Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (903) Google Scholar), are altered. Mice carrying a targeted mutation in Timp2 were generated. No Timp2 mRNA was detected in tissues from mutant animals, indicating the mutation was null. Loss of TIMP-2 did not adversely affect normal mouse development, viability, or fertility on the C57BL/6 background. However, loss of the inhibitor did result in a dramatic reduction in activation of the latent proenzyme, proMMP-2, in lung tissue in vivo and cultured primary fibroblastsin vitro. This demonstrates that TIMP-2 is not only a potential component of the proMMP-2 activation mechanism, as indicated by several published reports, but in fact it is required for normal activation of the latent proenzyme in vivo. It cannot be ruled out that TIMP-2-independent mechanisms exist for activating proMMP-2. However, if such mechanisms exist, they are far less efficient at inducing proMMP-2 activation than the mechanism involving TIMP-2 in the tissue and cells analyzed in this study. Failure of proMMP-2 activation in and of itself was expected to have minimal developmental consequences, consistent with the lack of a developmental phenotype in mice deficient for MMP-2 (50Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res. 1998; 58: 1048-1051PubMed Google Scholar). However, any phenotypes seen in MMP-2-deficient mice may also be expected to be seen in TIMP-2-deficient mice. Because the proMMP-2 activation defect observed in cultures ofTimp2 mutant cells could be suppressed by exogenously added recombinant human TIMP-2 protein, the requirement for TIMP-2 in proMMP-2 activation is not cell autonomous. The effects of added TIMP-2 protein on proMMP-2 activation in the culture systems used were either stimulatory or inhibitory, depending upon the concentration of TIMP-2 protein in the cultures. This is consistent with observations that the stoichiometry of the various co-activators and of the latent proMMP-2 protein is critical in governing proMMP-2 activation. Although exogenously added human TIMP-2 could partly restore proMMP-2 activation to homozygous mutant cells, the activation products that accumulated were intermediates and not fully active MMP-2. The reasons for the partial activation are not clear. There may be important species differences between mouse and human TIMP-2. The specific preparations of human TIMP-2 used may have been only partly active. Finally, the cellular location where TIMP-2 interacts with proMMP-2 may be important to the nature of the interaction and the capacity of proMMP-2 to subsequently undergo complete activation. Exogenously added TIMP-2 may not provide the same interaction with proMMP-2 as endogenously produced protein. TIMP-1 was unable to replace TIMP-2 as a co-factor for proMMP-2 activation; however, it was able to block proMMP-2 activation at higher concentrations. This indicated that MMP activity is itself needed for proMMP-2 activation and also highlights the functional differences between TIMPs 1 and 2. These differences are likely to result from the particular structural features of the two TIMPs. One specific feature that may allow TIMP-2, but not TIMP-1, to participate in proMMP-2 activation is the 10-residue-long negatively charged C-terminal tail of TIMP-2, which is only 3 residues long in TIMP-1 (41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). This tail may enable TIMP-2 to form salt bridges with specific positively charged residues at the junction of C-terminal hemopexin domains III and IV of pro-MMP-2, which are required for proMMP-2 to form a complex with TIMP-2 (37Butler 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 (538) Google Scholar, 51Overall 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). A second structural difference between TIMPs 1 and 2 that may enable TIMP-2 but not TIMP-1 to participate in proMMP-2 activation lies at the N terminus. Residues within the unique long N-terminal A-B β-hairpin loop of TIMP-2 are required for specific interactions with MMP-14 (52Butler G.S. Hutton M. Wattam B.A. Williamson R.A. Knauper V. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 20391-20396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). TIMP-1 is 7 residues shorter in this domain, which significantly shortens the A-B β-hairpin. This and other loop structure differences between the two TIMPs cause them to associate very differently with MMPs and may prevent TIMP-1 from interacting with MMP-14 in a way that enables it to replace TIMP-2 as a co-activator of proMMP-2 (41Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (307) Google Scholar). Activation mechanisms for proMMPs other than proMMP-2 have been described as cell surface events requiring membrane type MMPs. For example, like proMMP-2, proMMP-13 can be activated by a mechanism involving MMP-14 and MMP-2 (28Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). It is not yet known if efficient proMMP-13 activation, like proMMP-2 activation, is dependent upon the presence of TIMP-2. These studies are ongoing. Other TIMPs may also play a direct role in activation of proMMPs. TIMP-4 is able to interact with the C-terminal region of proMMP-2 in a manner similar to that of TIMP-2 (53Bigg H.F. Shi Y.E. Liu Y.E. Steffensen B. Overall C.M. J. Biol. Chem. 1997; 272: 15496-15500Crossref PubMed Scopus (143) Google Scholar). Whether it, like TIMP-2, is required for efficient proMMP-2 activation is not known. Other important functional differences between TIMPs 1 and 2 have been revealed during analysis of mutant mice. Although TIMP-2 loss resulted in defects in proMMP-2 activation, this was unaffected by a mutation within Timp1. Also, although TIMP-1-deficient animals are hyper-resistant to corneal infections with Pseudomonas aeruginosa by a complement-dependent mechanism, TIMP-2-deficient mice have normal immune responses (11Osiewicz K. McGarry M. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878: 494-496Crossref PubMed Scopus (13) Google Scholar, 54Wang Z. Soloway P.D. Ann. N. Y. Acad. Sci. 1999; 878 (21): 519Crossref PubMed Scopus (7) Google Scholar). It is possible, however, that other MMP-sensitive immune response mechanisms, possibly involving defensin production (55Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (903) Google Scholar), are altered. We are grateful to Drs. Hideaki Nagase and William Stetler-Stevenson for generously sharing recombinant TIMP proteins and Drs. Birkedal-Hansen and John Caterina for sharing cells and unpublished results.

Referência(s)