The founding member of the matrix metalloproteinase (MMP) family, collagenase, was identified in 1962 by Gross and Lapiere, who found that tadpole tails during metamorphosis contained an enzyme that could degrade fibrillar collagen1,2. Subsequently, an interstitial collagenase, collagenase-1 or MMP1, was found in diseased skin and synovium3. In vitro, MMP1 initiates degradation of native fibrillar collagens, crucial components of verte- brate extracellular matrix (ECM), by cleaving the peptide bond between Gly775���Ile776 or Gly775���Lys776 in native type I, II or III collagen molecules3,4. Further research led to the discovery of a family of structurally related proteinases (23 in human, 24 in mice), now referred to as the MMP family. Interest in MMPs increased in the late 1960s and early 1970s following observations that MMPs are upregulated in diverse human diseases including rheuma toid arthritis and cancer. Importantly, high levels of MMPs often correlated with poor prognosis in human patients (reviewed in REF. 5). However, recent clinical data indicate that the relationship between MMPs and disease is not simple for example, increased MMP activity can enhance tumour progression or can inhibit it (reviewed in REF. 6). This complex relationship between MMP expression and cancer has increased the basic and clinical interest in understanding MMP func- tion in vivo, but it has also focused attention on MMPs and pathology, and relatively less attention has been focused on the normal roles of these enzymes. Surely we do not have 23 MMPs in our bodies just to pro- mote tumours. A main question remains unanswered: what is the normal function of the MMP gene family in development? This Review focuses on what we have learned about the normal in vivo functions of MMPs from genetic analysis. MMPs in vivo: analysis of MMP mutants Analysis of genetic knockouts of MMPs offers opportun- ities to identify essential functions of an MMP and to validate candidate substrates by looking for cleavage products in the control animal and uncleaved proteins in the mutant animal. At least 14 mouse MMP mutants have been generated. The initial characterizations have described surprisingly subtle phenotypes, with all MMP- knockout lines surviving to birth (TABLE 1). Possible explan- ations for the seeming dispensability of MMPs during embryonic development include enzymatic redundancy, enzymatic compensation and adaptive development, although it is possible that MMPs are not important until after embryonic development. MMPs have many overlapping substrates in vitro5, which indicates possible genetic redun- dancy in vivo. Indeed, redundancy has now been shown with the recent generation of MMP double mutants7,8. Compensation has also been shown in the MMP family9,10. In the next sections, we will discuss mutant mouse pheno- types that rely on genetic approaches. Researchers can also now take advantage of simpler genetic model systems, such as Drosophila melanogaster, in which it is possible to mutate all MMP genes. MMP proteolysis MMPs are members of the metzincin group of proteases, which are named after the zinc ion and the conserved Met residue at the active site11,12. Recent work has generated a unified peptidase nomenclature13 in which MMPs include the M10A subfamily, the M10 family and the MA clan of metallopeptidases. Mammalian MMPs share a conserved domain structure (FIG. 1) that consists of a catalytic domain and an autoinhibitory pro-domain. The pro-domain contains a conserved Cys residue that coordin ates the active-site zinc to inhibit catalysis. *Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA. ���Department of Anatomy and Program in Biomedical Sciences, University of California, San Francisco, California 94143-0452, USA. ��These authors contributed equally to this paper. e-mails: pagema@rpi.edu andrew.ewald@ucsf.edu zena.werb@ucsf.edu doi:10.1038/nrm2125 Fibrillar collagen Polymerized, supramolecular collagen that has been organized into fibrils collagen types I, II and III form fibrils. Extracellular matrix Complex, ordered mixture of structural and signalling molecules that surrounds cells. Enzymatic redundancy Two enzymes that are expressed in the same time and place that can fully substitute for each other���s essential functions. Enzymatic compensation Upregulation of an enzyme, which is normally not expressed (or is expressed at a low level) to substitute for the absence of a mutated enzyme. Matrix metalloproteinases and the regulation of tissue remodelling Andrea Page-McCaw*��, Andrew J. Ewald����� and Zena Werb��� Abstract | Matrix metalloproteinases (MMPs) were discovered because of their role in amphibian metamorphosis, yet they have attracted more attention because of their roles in disease. Despite intensive scrutiny in vitro, in cell culture and in animal models, the normal physiological roles of these extracellular proteases have been elusive. Recent studies in mice and flies point to essential roles of MMPs as mediators of change and physical adaptation in tissues, whether developmentally regulated, environmentally induced or disease associated. REVIEWS NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | MARCH 2007 | 221 �� 2007 Nature Publishing Group

Adaptive development Alternative developmental trajectory whereby an organism compensates for the loss of a gene by doing some essential function in a reproducibly different manner. Catalytic domain A domain that cleaves other proteins. Pro-domain An autoinhibitory domain that prevents the catalytic domain from functioning. When the pro-domain is destabilized or removed, the active site becomes available to cleave substrates. Most MMP- family members also contain a hemopexin domain, attached at their C termini by a flexible hinge. The hemo pexin domain encodes a four-bladed ��-propeller structure that mediates protein���protein interactions. This domain also contributes to proper substrate recognition, activation of the enzyme, protease localization, inter nalization and degradation14. The structures of the catalytic and hemopexin domains of several MMPs, including MMP1, MMP2, MMP3 and MMP14 (also known as mem- brane type 1 MMP (MT1-MMP)), have been solved (reviewed in REFS 13,15). MMP2 and MMP9 also have fibronectin type II repeats, which mediate binding to collagens, inserted into the catalytic domain. Most MMPs are secreted proteins however there are six MT-MMPs: MMP14, MMP15, MMP16 and MMP24 have transmembrane domains and short cytoplasmic tails MMP17 and MMP25 have glycosylphosphatidylinositol (GPI) linkages. Also, MMP23 is a type II transmembrane protein. The activity of MMPs is controlled at many levels and the regulation of MMP activity remains a topic of intense research (BOX 1). Functions of MMP proteolysis. Historically, MMPs were thought to function mainly as enzymes that degrade structural components of the ECM. However, MMP pro- teolysis can create space for cells to migrate, can produce specific substrate-cleavage fragments with independent biological activity, can regulate tissue architecture through effects on the ECM and intercellular junctions, and can activate, deactivate or modify the activity of sig- nalling molecules, both directly and indirectly16 (FIG. 2). Because cells have receptors (for example, integrins) for structural ECM components, MMPs can also affect cellu- lar functions by regulating the ECM proteins with which the cells interact17. In many cases, MMP cleavage of ECM substrates generates fragments that have different biological activities from their precursors. For example, the cleavage of laminin-5 or collagen IV results in the exposure of cryptic sites that promote migration18,19. Type I collagen degradation that is mediated by MMP1 is necessary for epithelial cell migration and wound healing in culture models20. Cleavage of ECM proteins by MMPs can also release ECM-bound growth factors, including insulin growth factors and fibroblast growth factors21,22. Alternative mechanisms of action have also been observed, including functional intermolecular MMP complexes: MMP14 binds to tissue inhibitor of metalloproteinase-2 (TIMP2), which binds to pro-MMP2, thereby positioning it for activation by a second molecule of MMP14 (REF. 23). Furthermore, human MMP11 has an alternative splice isoform that functions as an intracellular proteinase and enters the nucleus24. MMP substrates include peptide growth factors, tyro- sine kinase receptors, cell-adhesion molecules, cytokines and chemokines, as well as other MMPs and unrelated proteases (BOX 2). MMPs and the related families of pro- teinases, the ADAMs (a disintegrin and metallo proteases) and ADAM-TSs (ADAMs with thrombo spondin repeats), are important in shedding plasma-membrane- bound proteins. ADAMs and ADAM-TSs participate in shedding growth factors that are synthesized as cell-membrane-bound precursor forms, including heparin-binding epidermal growth factor (HB-EGF), neuregulin, amphiregulin and transforming growth factor-�� (TGF��)25���28. Cleavage of other membrane proteins, such as E-cadherin and CD44, results in the release of specific, biologically active fragments of their extra cellular domains, and in increased invasive behaviour29,30. Cell-surface-adhesion molecules, such as syndecan-1, are also shed by soluble and membrane MMPs31,32. MMP9 and MMP12 contribute to proteo- lytic shedding of the lipopolysaccharide (LPS) receptor CD14, and therefore influence innate host defense33. Table 1 | Selected MMP- and TIMP-null mutant phenotypes MMP gene Mutant phenotypes (mouse, unless noted) Mmp2 Reduced body size152 reduced neovascularization55 decreased primary ductal invasion in the mammary gland54 reduced lung saccular development153 Mmp3 Altered structure of neuromuscular junctions154 reduced purse stringing during wound healing86 altered secondary branching morphogenesis in the mammary gland54 Mmp7 Innate immunity defects83 decreased re-epithelialization after lung injury85 Mmp8 Increased skin tumours104 resistance to tumour necrosis factor (TNF)- induced lethal hepatitis103 Mmp9 Bone-development defects36 defective neuronal remyelination after nerve injury155 delayed healing of bone fractures39 impaired vascular remodelling156 impaired angiogenesis36 Mmp10 Increased inflammation and increased mortality in response to infection or wounding (W. C. Parks , personal communication) Mmp11 Delayed mammary tumorigenesis157 Mmp12 Diminished recovery from spinal cord crush158 increased angiogenesis due to decreased angiostatin128 Mmp13 Bone remodelling defects7, 40 reduced hepatic fibrosis159 increased collagen accumulation in atherosclerotic plaques160 Mmp14 Skeletal remodelling defects42, 43, 45, 161 angiogenesis defects46 inhibition of tooth eruption and root elongation162 defects in lung and submandibular gland46, 163 Mmp19 Obesity164 Mmp20 Defects in tooth enamel165 Mmp23 No phenotype reported Mmp24 Abnormal response to sciatic nerve injury166 Mmp28 Increased inflammatory response (W. C. Parks , personal communication) DmMmp1 Defective tracheal tube growth (see text)75 failure of head eversion at metamorphosis75 DmMmp2 Failure of tissue histolysis at metamorphosis75 Timp1 Accelerated endometrial gland formation167 impaired learning and memory168 accelerated hepatocyte cell-cycle progression169 increased resistance to Pseudomonas infection107 Timp2 Motor defects170 Timp3 Accelerated apoptosis in mammary glands171 impaired bronchiole branching172 enhanced metastatic dissemination173 Timp4 No phenotype reported174 DmTimp Inflated wings175 autodigesting gut175 Dm, Drosophila melanogaster Mmp, matrix metalloproteinase TIMP, tissue inhibitor of metalloproteinase. REVIEWS 222 | MARCH 2007 | VOLUME 8 www.nature.com/reviews/molcellbio �� 2007 Nature Publishing Group