Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme ( ABSTRACTCertain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

We report that matrilysin, a matrix metalloproteinase, is constitutively expressed in the epithelium of peribronchial glands and conducting airways in normal lung. Matrilysin expression was increased in airway epithelial cells and was induced in alveolar type II cells in cystic fibrosis. Other metalloproteinases (collagenase-1, stromelysin-1, and 92-kD gelatinase) were not produced by normal or injured lung epithelium. These observations suggest that matrilysin functions in injury-mediated responses of the lung. Indeed, matrilysin expression was increased in migrating airway epithelial cells in wounded human and mouse trachea. In human tissue, epithelial migration was reduced by Ͼ 80% by a hydroxamate inhibitor, and in mouse tissue, reepithelialization in trachea from matrilysin-null mice was essentially blocked. In vivo observations and cell culture studies demonstrated that matrilysin was secreted lumenally by lung epithelium, but upon activation or while migrating over wounds, some matrilysin was released basally. The constitutive production of matrilysin in conducting airways, its upregulation after injury, its induction by alveolar epithelium, and its release into both lumenal and matrix compartments suggest that this metalloproteinase serves multiple functions in intact and injured lung, one of which is to facilitate reepithelialization. ( J. Clin. Invest. 1998. 102:1321-1331.)

We have cloned a new human matrix metalloproteinase (MMP-28, epilysin) from human keratinocyte and testis cDNA libraries. Like most MMPs, epilysin contains a signal sequence, a prodomain with a PRCGVTD sequence, a zinc-binding catalytic domain with an HEIGHTLGLTH sequence, and a hemopexin-like domain. In addition, epilysin has a furin activation sequence (RRKKR) but has no transmembrane sequence. The exon-intron organization and splicing pattern of epilysin differ from that of other MMP genes. It has only 8 exons, and 5 exons are spliced at sites not used by other MMPs. Another novel feature of epilysin is that exon 4 is alternatively spliced to a transcript that does not encode the N-terminal half of the catalytic domain. Northern hybridization of tissue RNA indicated that epilysin is expressed at high levels in testis and at lower levels in lungs, heart, colon, intestine, and brain. RNase protection assay with various cell lines indicated that epilysin was selectively expressed in keratinocytes. Recombinant epilysin degraded casein in a zymography assay, and its proteolytic activity was inhibited by EDTA and by batimastat, a selective MMP inhibitor. Immunohistochemical staining showed expression of epilysin protein in the basal and suprabasal epidermis of intact skin. In injured skin, prominent staining for epilysin was seen in basal keratinocytes both at and some distance from the wound edge, a pattern that is quite distinct from that of other MMPs expressed during tissue repair. These findings suggest that this new MMP functions in several tissues both in tissue homeostasis and in repair. The matrix metalloproteinases (MMPs)1 compose a family of enzymes that share several common structural features and that function both in the turnover and degradation of extracellular matrix proteins and in the processing, activation, or deactivation of a variety of soluble factors (1). MMPs, or matrixins, are a subgroup of the much larger metalloproteinase superfamily, which also includes astacin and ADAM proteinases, among others. To date 23 different MMPs have been cloned, and additional members continue to be identified (2).To be classified as a matrix metalloproteinase, a protein must have conserved features of two domains, namely the prodomain and the catalytic domain. The prodomain of a typical MMP is about 80 amino acids, and all MMPs, except MMP-23 (3), contain the consensus sequence PRCXXPD. As for all metalloproteinases, the catalytic domain contains an active site Zn 2ϩ that binds three conserved histidines in the sequence HEXXHXXGXXH(S/T)XXXXXXM, which also contains a conserved methionine to the carboxyl side of the zinc-binding site (metzincins) (4). In an inactive state, the conserved cysteine residue in the prodomain provides the fourth coordination site for the catalytic zinc ion. In addition, with the exception of matrilysin (MMP-7), endometase/matrilysin-2 (MMP-26), and MMP-23, MMPs have a hinge region, which is often prolinerich, and a so-called hemopexin-like C-terminal domain (3,5,6). Other domains fou...

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the ␣ 2 ␤ 1 integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of ␣ 2 ␤ 1 contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with ␣ 2 ␤ 1 from keratinocytes, and ␣ 2 ␤ 1 co-immunoprecipitated with pro-MMP-1. No other MMPs bound ␣ 2 ␤ 1 , and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for ␣ 2 ␤ 1 -dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the ␣ 2 I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with ␣ 2 ␤ 1 confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.Cells, either resting or activated, use a variety of surface receptors to sense the presence and location of specific molecules in the extracellular space. For example, integrin-ligand interactions tell cells which structural proteins they have encountered in the extracellular space, and in turn, these contacts activate signaling pathways involved in differentiation, proliferation, and gene expression, among other processes. During migration, cells need to proteolyze, to some extent, nearby extracellular matrix proteins, and hence, cell-matrix contacts can also instruct cells which proteinases are needed and where the enzyme should be targeted and released.An example of cell-matrix-mediated spatial regulation of proteolysis is seen with collagenase-1 (MMP-1), 1 a matrix metalloproteinase, in human cutaneous wounds. In response to injury, collagenase-1 is induced in basal epidermal cells (keratinocytes) as the cells move off of the basement membrane and contact native type I collagen in the underlying dermis (1), and this inductive response is specifically controlled by the collagen-binding integrin ␣ 2 ␤ 1 (2). As we demonstrated in various experiments, the catalytic activity of collagenase-1 is required and sufficient for keratinocyte migration on complex matrices containing type I collagen. For example, keratinocytes plated on mutant, collagenase-resistant type I collagen do not migrate, even in the presence of fibronectin and vitronectin; yet they express MMP-1 at levels equivalent to those released by cells on wild-type collagen (2). Keratinocyte migration is also completely inhibited by anti-collagenase-1 antibodies, which block the catalytic a...

Matrix metalloproteinases (MMPs) are a family of inducible enzymes that degrade extracellular matrix components, allowing cells to traverse connective tissue structures efficiently. Specific tissue inhibitors (TIMPs) function as physiologic inhibitors of MMP activity. Because neovascularization may require various proteinases, we characterized the profile of metalloenzyme production by microvascular endothelial cells (MEC) and the modulation of expression by phorbol esters (PMA) and by the physiologically relevant cytokines tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor, and interferon-gamma. MMP expression by MEC and large-vessel human umbilical vein endothelial cells (HUVEC) was determined by enzyme-linked immunosorbent assay, immunoprecipitation, Northern hybridization, and transfection assays. Constitutive expression of MMPs by endothelial cells was low. PMA stimulated the production of collagenase, stromelysin, 92-kDa gelatinase, and TIMP-1 in both endothelial cell types. TIMP-2 was constitutively expressed by MEC and HUVEC, but was down-regulated by PMA. TNF-alpha induced an endothelial-cell-specific up-regulation of collagenase with a concomitant inhibition of PMA-induced TIMP-1 up-regulation, a response that is distinct from that of fibroblasts. Interferon-gamma up-regulated TIMP-1 production by MEC and blocked PMA and TNF-induced up-regulation of collagenase. Northern hybridization assays showed pretranslational control of PMA-, basic fibroblast growth factor-, and TNF-alpha-induced MMP expression. Collagenase-promoter CAT constructs containing 2.28 kb of the 5' region of the collagenase gene demonstrated transcriptional regulation. The potential physiologic relevance of such regulation was shown in an in vitro migration assay. MEC were stimulated to migrate by wounding and exposure to TNF-alpha. Collagenase mRNA was prominently expressed by the migrating cells, as shown by in situ hybridization. In sum, MEC have a unique profile of MMP expression and regulation compared with other cell types, which may be important for wound healing and angiogenesis, particularly during the early phase of migration.