Title: ECM and Cell Surface Proteolysis: Regulating Cellular Ecology
Abstract: Cell–cell and cell–extracellular matrix (ECM) interactions provide cells with information essential for controlling morphogenesis, cell fate specification, gain or loss of tissue-specific functions, cell migration, tissue repair, and cell death. Degradation or activation of cell surface and ECM proteins by proteolysis can mediate rapid and irreversible responses to changes in the cellular microenvironment. By concentrating proteolytic events at or near the cell surface, these processes can be effective even in the presence of high concentrations of inhibitors. This minireview considers the cellular and organismal functions of ECM proteinases. Their biochemical and structural properties are reviewed elsewhere (14Stocker W Grams F Baumann U Reinemer P Gomis-Ruth F.-X McKay D.B Bode W Protein Sci. 1995; 4: 823-840Crossref PubMed Scopus (634) Google Scholar, 19Wolfsberg T.G White J.M Dev. Biol. 1996; 180: 389-401Crossref PubMed Scopus (216) Google Scholar, 1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar, 13Sternlicht, M., and Werb, Z. (1997). In Guidebook to the Extracellular Matrix and Adhesion Proteins, 2nd Ed., T. Kreis and R. Vale, eds. (Oxford: Oxford University Press), in press.Google Scholar). Proteolysis regulates ECM assembly, editing of excess ECM components, remodeling of ECM structure, and release of bioactive fragments and growth factors during growth, morphogenesis, tissue repair, and pathological processes. The major enzymes that degrade ECM and cell surface proteins (Table 1) are the matrix metalloproteinase (MMP) family of secreted and membrane proteinases, the adamalysin-related membrane proteinases that contain disintegrin and metalloproteinase domains (ADAMs or MDCs), the bone morphogenetic protein 1 (BMP1)/tolloid (tld) family of metalloproteinases, and tissue serine proteinases, such as thrombin, tissue plasminogen activator (tPA), urokinase (uPA), and plasmin.Table 1ECM and Cell Surface Substrates of Pericellular ProteinasesEnzymesOther NamesSubstratesaSubstrates are grouped for similar enzymes, but not all of the proteins in each group are cleaved by all the enzymes in the group.MMPsMMP-1 MMP-8 MMP-13collagenase-1 collagenase-2 collagenase-3Col I, II, III, VII, X, GL, EN, LP, AG, TN, L-selectin, IGF-BP, Pro-MMP-2, -9, α2M, α1PIMMP-2 MMP-9gelatinase A gelatinase BGL, Col I, IV, V, VII, X, XI, EL, FN, LN, LP, AG, galectin-3, IGF-BP, VN, FGF receptor-1, Pro-MMP-2, -9, -13MMP-3 MMP-10 MMP-7stromelysin-1 stromelysin-2 matrilysinPG, LN, FN, GL, Col III, IV, V, IX, X, XI, LP, FB, EN, TN, VN, Pro-MMP-1, -8, -9, -13, α1PI, α2M, L-selectinMMP-12metalloelastaseEL, FB, FN, LN, PG, MBP, PL, α1PIMMP-14 MMP-15MT1-MMP MT2-MMPCol I, II, III, GL, FN, LN, VN, PG, Pro-MMP-2, -13, α1PI, α2MMMP-11stromelysin-3LN, FN, AG, α1PI, α2MOther ProteinasesuPAPL, FN, HGFtPAPlasminFB, FN, TN, LN, AG, latent TGFβ BP, PG, Pro-MMP-1, -3, -9, -14, C1, C3, C5ThrombinFB, Pro-MMP-2, syndecanBMP-1tld (procollagen C-peptidase)proCOL I, LN-5, latent TGFβ familyKuzbanianADAM 10 (Kuz)Notch, cell-bound TNFα, MBPTACEADAM 17cell bound TNFαAbbreviations: aggrecan, AG; α1-proteinase inhibitor, α1PI; α2-macroglobulin, α2M; binding protein, BP; collagen type, Col; complement, C; elastin, EL; entactin, EN; fibrin/fibrinogen, FB; fibronectin, FN; gelatins, GL; hepatocyte growth factor, HGF; laminin, LN; link protein, LP; myelin basic protein, MBP; plasminogen, PL; proteoglycans, PG; tenascin, TN; vitronectin, VN; gelatinase A, 72 kDa gelatinase; gelatinase B, 92 kDa gelatinase.a Substrates are grouped for similar enzymes, but not all of the proteins in each group are cleaved by all the enzymes in the group. Open table in a new tab Abbreviations: aggrecan, AG; α1-proteinase inhibitor, α1PI; α2-macroglobulin, α2M; binding protein, BP; collagen type, Col; complement, C; elastin, EL; entactin, EN; fibrin/fibrinogen, FB; fibronectin, FN; gelatins, GL; hepatocyte growth factor, HGF; laminin, LN; link protein, LP; myelin basic protein, MBP; plasminogen, PL; proteoglycans, PG; tenascin, TN; vitronectin, VN; gelatinase A, 72 kDa gelatinase; gelatinase B, 92 kDa gelatinase. ECM degradation in vivo is confined to the immediate pericellular environment of cells. Antibodies that recognize neoepitopes created by proteolytic cleavage show staining concentrated around cells, even when the proteinase is soluble. But how can the proteinases be used in a spatially confined manner? The proteolytic events are exquisitely regulated and confined by localizing the enzymes to receptors, adhesion sites, or invasive protrusions of cells where ECM degradation takes place (1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar, 10Nakahara H Howard L Thompson E.W Sato H Seiki M Yeh Y Chen W.-T Proc. Nat. Acad. Sci. USA. 1997; 94: 7959-7964Crossref PubMed Scopus (363) Google Scholarreferences therein). Several distinct mechanisms are used to target proteinases to specific membrane domains. A sequence in the transmembrane/cytoplasmic domain of MT1-MMP targets it to invasive sites. Not only is MT1-MMP an ECM-degrading proteinase in its own right, but, in a complex with the tissue inhibitor of metalloproteinases (TIMP)-2, it is also an activator and receptor for gelatinase A (Figure 1). A different strategy is used to confine uPA to the uPA receptor (uPA-R) at the invasive edge of migrating cells. uPA-R is itself an adhesion receptor for vitronectin, but also interacts laterally with integrin β chains. Regulating the localization of membrane-bound proteinases to membrane domains proximal to their substrates by interactions with the cytoskeleton would modulate proteolysis rapidly. Such a mechanism could explain how the shedding of L-selectin and syndecan can occur within minutes upon cell stimulation. Through these diverse mechanisms, adhesion, deadhesion, and ECM proteolysis are all brought to bear at the leading edge of migrating and invading cells where dynamic changes take place. However, the ECM, per se, is not the only target of pericellular proteolysis (Table 1). During the cellular response to developmental and pathologic cues, cell surface proteins, receptors, and transmembrane ECM proteins are altered by proteolysis. The nature of the proteinases mediating these events has been elusive, but there are now emerging concepts supporting a role for integral membrane proteinases. The ADAMs (19Wolfsberg T.G White J.M Dev. Biol. 1996; 180: 389-401Crossref PubMed Scopus (216) Google Scholar, 2Blobel C Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) are particularly intriguing because they contain both cell adhesion and proteolytic domains. Kuz, the Drosophila homolog of ADAM 10, activates the signaling receptor Notch during development and TNFα converting enzyme (TACE) releases membrane-bound TNFα by proteolysis. Genetic modification of proteinase and inhibitor gene expression in vivo is necessary to elucidate their real functions (5Coussens L.M Werb Z Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar, 12Shapiro S.D Matrix Biol. 1997; 15: 527-533Crossref PubMed Scopus (43) Google Scholar). The initial studies of null mutants of uPA, tPA, MMPs, and their inhibitors PAI-1 and TIMP-1 were disappointing because of their mild phenotypes. In contrast, their ectopic expression produces pathologic remodeling. This suggests that requirements for pericellular proteolysis become evident only under conditions of acute perturbation. What is less obvious is why lack of specific proteinases produces so few defects during development. Perhaps, under conditions of rapid growth with the abundant synthesis of new ECM molecules, the degradation of a much smaller amount of ECM left behind during migration or branching is irrelevant. However, deficient proteolysis leads to disease processes, just as overproduction of proteinases does. In vivo, degradation of the provisional ECM produced by wounding is a key step in the healing process. Once the fibrin clot has formed, migrating keratinocytes, which produce MMPs and uPA, close the wound surface and interact with the underlying dermal fibroblasts to repair the injury. In mice that have a targeted null mutation in their plasminogen gene, keratinocytes at the wound edge do not degrade fibrin and are incapable of migrating, and the wound does not heal (3Bugge T.H Kombrinck K.W Flick M.J Daugherty C.C Danton M.J Degen J.L Cell. 1996; 87: 709-719Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholarreferences therein). Keratinocyte behavior is particularly sensitive to ECM fragments produced by proteolysis. Cleavage of epithelial laminin-5 by gelatinase A induces keratinocyte migration (6Giannelli G Falk-Marzillier J Stetler-Stevenson W.G Schiraldi I Quaranta V Science. 1997; 277: 225-228Crossref PubMed Scopus (1041) Google Scholar), whereas keratinocytes are unable to migrate on collagen type I that has been rendered resistant to proteolysis by mutating the collagenase cleavage site (11Pilcher B.K Dumin J.A Sudbeck B.D Krane S.M Welgus H.G Parks W.C J. Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (494) Google Scholar). Moreover, a decrease in stromelysin-1 expression owing to a promoter polymorphism is associated with increased progression of a common human disease, atherosclerosis (20Ye E Eriksson P Hamsten A Kurkinen M Humphries S.E Henney A.M J. Biol. Chem. 1996; 271: 13055-13060Crossref PubMed Scopus (439) Google Scholar). These studies support the need for pericellular proteolysis in migration and remodeling. The enzymes that cleave the N- and C-terminal propeptides of the fibrillar collagens during collagen assembly have long been sought. The surprising discovery that BMP1 cleaves the C-terminal propeptide of type I collagen supports the original finding that a metalloenzyme is involved. BMP1, which was isolated as an inducer of bone formation, is related to tld, which activates decapentaplegic, a member of the TGFβ superfamily that specifies the dorsal–ventral axis in Drosophila. Mouse embryos with homozygous deletions in the BMP1/tld gene develop until late gestation with few morphologic defects, but have abnormal collagen fibrils and die at birth (15Suzuki N Labosky P.A Furuta Y Hargett L Dunn R Fogo A.B Takahara K Petero D.M Greenspan D.S Hogan B.L Development. 1996; 122: 3587-3589Crossref PubMed Google Scholarreferences therein). However, mutations in tld in Drosophila have severe developmental consequences. This opens up the possibility that ECM proteinases important in mammalian embryogenesis still remain to be discovered. ECM provides adhesive signals that control cell viability. Degradation of ECM is the most effective mechanism for altering anchorage in vivo. Controlling apoptosis by regulating ECM proteolysis is likely to be widespread in epithelial tissues, which require adhesion for survival. It may be the loss of responsiveness to this feature of ECM signaling that underlies immortalization, the first, critical step toward neoplasia. In the mammary gland, ECM is remodeled during the differentiation cycle of pregnancy, lactation, and involution (5Coussens L.M Werb Z Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (503) Google Scholar). Proteinases mediate ECM degradation, in parallel with apoptosis during involution after lactation. Ectopic expression of stromelysin-1 induces unscheduled entactin degradation and mammary involution, whereas its inhibition delays apoptosis. What is particularly fascinating is that the same MMP that induces apoptosis during pregnancy, stimulates ductal branching morphogenesis during puberty. Specificity may come from using different ECM targets or receptors at different times in development. Programmed cell death also regulates neuronal communication, both during development and in disease processes. In the hippocampus tPA participates in neuronal plasticity. Plasmin generated by tPA induces neuronal cell death (16Tsirka S.E Rogove A.D Bugge T.H Degen J.L Strickland S J. Neurosci. 1997; 17: 543-552Crossref PubMed Google Scholarreferences therein), probably through degradation of ECM molecules that are essential for neuronal survival in culture. The excitotoxin-stimulated neurons no longer die in animals null for tPA. Interestingly, in both mammary gland and brain, the proteinases are expressed by the supporting cells (stromal or microglial), not by the epithelial or neuronal cells that die. Thus, apoptosis is an example of cell-to-cell communication, with ECM proteinases as the mediators. However, the cleavage of ECM components can also enhance viability of cells by the generation of bioactive fragments of ECM proteins or release of cytokines. In the case for melanoma cells, degradation of collagen promotes survival by liberating fragments recognized by αvβ3 integrin (17Varner J.A Cheresh D.A Curr. Opin. Cell Biol. 1996; 8: 724-730Crossref PubMed Scopus (468) Google Scholar). ECM-degrading MMPs, uPA, and other proteinases are universally expressed during tumor progression and metastasis (5Coussens L.M Werb Z Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar). The majority of proteinases are made by the responding stroma of epithelial tumors. The enzymes can then bind to the surface of tumor cells. Whether or how these proteinases are involved in angiogenesis, tumor initiation, growth, metastasis, or in the host defense against the tumor remains to be determined. Most commonly, proteinases facilitate tumor progression. Proteinases may act on ECM to free cells from ECM-induced cell cycle arrest, facilitate dedifferentiation, mediate invasion, or release latent growth/angiogenesis factors (5Coussens L.M Werb Z Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (503) Google Scholar). Blocking function of ECM proteinases can decrease tumor growth (9Martin D.C Ruther U Sanchez-Sweatman O.H Orr F.W Khokha R Oncogene. 1997; 13: 569-576Google Scholar, 18Wilson C.L Heppner K.J Labosky P.A Hogan B.L Matrisian L.M Proc. Natl. Acad. Sci. USA. 1997; 94: 1402-1407Crossref PubMed Scopus (546) Google Scholar), while their ectopic expression can increase carcinogenic potential, indicating that they contribute to the neoplastic process. However, proteinases also may inhibit tumorigenesis by inducing apoptosis or by releasing latent angiogenesis inhibitors from ECM proteins. One overlooked aspect of pericellular proteolysis is its potential role in immunity and host defense. ECM proteolytic cascades resemble those of complement activation and coagulation, and macrophages and other inflammatory cells involved in the innate immune response express many ECM proteinases (1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar, 12Shapiro S.D Matrix Biol. 1997; 15: 527-533Crossref PubMed Scopus (43) Google Scholar). That extracellular proteinases regulate host defense through effects on migration or processing of cytokines, complement, or fibrin, is suggested by decreased response of macrophages to inflammatory stimuli in metalloelastase-deficient mice and increased susceptibility of uPA-deficient mice to infection. Moreover, matrilysin, which is secreted luminally in epithelia, is critically placed to activate the antimicrobial cryptidins. TNFα, a potent mediator of inflammation, is synthesized as a transmembrane molecule that can bind to its receptor by cell–cell contact. It is processed by proteolysis to a soluble homotrimer by TACE (2Blobel C Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Shedding of other cell surface molecules involved in innate immunity, including the adhesion molecule L-selectin, the apoptosis inducer Fas ligand, TGFα, and the IL-6 receptor, is mediated by enzymes that may be distinct from TACE. ECM proteinases may be as important as regulators of cytokine function, epithelial barrier function, or antimicrobial activity, as they are in ECM proteolysis. During cellular responses to developmental and pathologic cues, ECM, cell surface proteins, and receptors are activated or removed by proteolysis. Just as protein modification by phosphorylation is an effective means of intracellular signal transduction, protein modification by proteolysis, by virtue of its irreversible nature, is ideally suited to regulate extracellular signal transduction in the pericellular environment and thus to push forward the unidirectional decisions of development and disease (Figure 2). The prototype for such signaling is the protease-activated receptor (PAR) family, which encode blocked, tethered ligands (7Ishihara H Connolly A.J Zeng D Kahn M.L Zheng Y.W Timmons C Tram T Coughlin S.R Nature. 1997; 386: 502-506Crossref PubMed Scopus (798) Google Scholarreferences therein). Thrombin cleaves PAR-1 or PAR-3, initiating a G protein–coupled cascade that can regulate fibroblast proliferation or neuronal survival. PAR-2 activation by tryptase released by mast cells may underlie increased collagen I synthesis by fibroblasts (4Cairns J.A Walls A.F J. Clin. Invest. 1997; 99: 1313-1321Crossref PubMed Scopus (268) Google Scholarreferences therein) seen in fibrosis, inflammation, and neoplastic progression. The binding of uPA to uPA-R is a distinct paradigm. uPA induces interaction of uPA-R with β1 and β2 integrins, thereby regulating adhesion and signaling. Although bound uPA is active, its activity is not required for signaling (1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar). Whether similar signaling takes place for other interactions of MMPs or ADAMs with their receptors remains to be determined. ADAMs, which can process or remove the extracellular domains of cell surface proteins, are critically placed for regulating signaling (2Blobel C Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Indeed, Kuz processes Notch so that it is competent for signaling in development. It is intriguing that the extracellular domain of Notch 4 is deleted in the constitutively active, oncogenic form that induces breast cancer. Proteolysis of extracellular domains of tumor suppressors, such as Notch 4, E-cadherin, syndecan, or DCC (deleted in colon carcinoma), would alter their signaling functions phenotypically. Such ECM proteinases could have major consequences for tumorigenesis. Proteolysis also releases cryptic activities of ECM ligands that may signal differently than the intact protein. Once cleaved, ECM fragments may bind different integrins; e.g., αvβ3 for cleaved collagen, versus α1β2 for intact collagen. Engaging only the central cell-binding domain of fibronectin activates AP-1, while whole fibronectin does not, and cleaved laminin-5 activates different pathways than the intact molecule (6Giannelli G Falk-Marzillier J Stetler-Stevenson W.G Schiraldi I Quaranta V Science. 1997; 277: 225-228Crossref PubMed Scopus (1041) Google Scholarreferences therein). Sequestration, presentation, or activation of growth factors is also regulated by proteolysis. Latent TGFβ- and IGF-binding proteins are ECM proteins that can be degraded by proteinases, releasing the growth factors. BMP1/tld may activate TGFβ family proteins by cleaving latency proteins, whereas uPA cleaves the HGF precursor to its active form. In contrast, with degradation of heparan sulfate proteoglycans, which are coreceptors for FGF, Wnt, and VEGF family members, the sequestration of growth factors next to their receptors is lost and growth factor signaling is compromised (1Andreasen P.A Kjoller L Christensen L Duffy M.J Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar, 8Kispert A Vainio S Shen L Rowitch D.H McMahon A.P Development. 1996; 122: 3627-3637Crossref PubMed Google Scholarreferences therein). Our understanding of proteolysis of ECM and cell surface molecules has expanded dramatically. We now know that the most significant proteolytic events are confined to the pericellular environment. With the discovery of new proteinases with new functions come new avenues for exploration in areas of development and disease. The synergy between adhesion, ECM, and proteolysis has been advanced by discovery of the intimate relationships between proteinases and signal transduction, particularly as effectors of epithelial–stromal interactions. Understanding the mechanisms by which pericellular proteinases are regulated and activated, the nature of their molecular targets, and how adhesion and proteolysis are integrated will provide exciting avenues for investigation over the next few years.