Susan S. Yamada

MT1-MMP-Deficient Mice Develop Dwarfism, Osteopenia, Arthritis, and Connective Tissue Disease due to Inadequate Collagen Turnover

MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Electron microscopy and structural model of human fibronectin receptor.

Highly‐purified human fibronectin receptor (a heterodimer of two distinct subunits, alpha and beta) was studied using electron microscopy and a variety of preparative procedures. It was found that the receptor consists of a globular head approximately 80 by 120 A and two tails about 20 A thick and 180‐200 A long. The whole complex is approximately 280 A long. At low concentrations of detergent the receptor forms doublets, triplets or rosettes associated with the tails which possess the transmembrane portion of the molecule. Computer‐assisted structure prediction using the published amino acid sequence of both subunits showed differences in the secondary structure of the tails, the alpha‐tail being rich in beta‐strands, the beta‐tail having five cysteine‐rich repeats analogous to the EGF‐like repeats of laminin. Estimates of the length of the tails from the predicted structure conformed well with the dimensions obtained from electron micrographs.

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin: variable expression in basal and squamous cell carcinomas

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts

The distribution of microfilament bundles in cells was examined using antibodies to fibroblast myosin and indirect immunofluorescence microscopy. There is no correlation between the presence of bundles of microfilaments and normal growth control. A normal cell line (Balb/c 3T3) cultured on a poorly adhesive substratum showed no microfilament bundles. Similarly, a mutant cell line (AD6) with normal growth, but a rounded shape due to defective adhesiveness to substratum, showed no bundle formation. On the other hand, two transformed cell lines with a flat morphology (Swiss SV3T3 and Balb MSV-85) showed extensive bundle formation. When a transformed cell line with poor adhesiveness (MC5-5) was treated with CSP (a major surface glycoprotein of normal cells) which increases adhesiveness to substratum, the cells formed extensive microfilament bundles without any decrease in growth. We conclude that the distribution of microfilament bundles is related to adhesiveness to substratum and cell shape but not to growth properties.

MT1-MMP: A tethered collagenase†

Gene ablation in mice offers a powerful tool to assay in vivo the role of selected molecules. Numerous new mouse models of matrix metalloproteinases (MMP) deficiency have been developed in the past 5 years and have yielded a new understanding of the role of MMPs while also putting to rest assumptions based on data predating the days of mouse models. The phenotype of the MT1‐MMP deficient mouse is one example which illustrates the sometimes rather surprising insights into extracellular matrix remodeling in development and growth that can be gained with mouse genetics. While MT1‐MMP appears to play little or no role in embryonic development, loss of this enzyme results in progressive impairment of postnatal growth and development affecting both the skeleton and the soft connective tissues. The underlying pathologic mechanism is loss of an indispensable collagenolytic activity, which remains essentially uncompensated. Our findings demonstrate that growth and maintenance of the skeleton requires coordinated and simultaneous MT1‐MMP‐dependent remodeling of all soft tissue attachments (ligaments, tendons, joint capsules). We note that the phenotype of the MT1‐MMP deficient mouse bears no resemblance to those of mice deficient in MMP‐2 and tissue inhibitors of metallo‐proteinase (TIMP)‐2 all but dispelling the view that activation of MMP‐2 by the MT1‐MMP/TIMP‐2/proMMP‐2 axis plays a significant role in growth and development throughout life. It is of interest to note that loss of a single catabolic function such as selective collagen degradation mediated by MT1‐MMP gives rise to profound impairment of a number of both anabolic and catabolic functions.

The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

MT1-MMP–dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)–dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of “ghost” cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

Mannose receptor–mediated uptake of collagen by M2-like macrophages is a major mechanism of collagen turnover in mice.

Membrane-type 1 matrix metalloproteinase is required for normal alveolar development.

Matrix metalloproteinases (MMPs) are expressed during lung development, but their role may be limited, as mice deficient in MMP‐3, 7, 9, or 12 develop a normal adult lung. Because membrane‐type 1 matrix metalloproteinase (MT1‐MMP) is expressed in the developing lung epithelium, we examined the lung structure of MT1‐MMP–deficient (−/−) mice. Branching morphogenesis was normal, but alveolar development was abnormal in the MT1‐MMP−/− lungs with 40% less alveolar surface area at 1 month (P < 0.01). MT1‐MMP−/− airways and alveoli had an abnormal ultrastructural appearance, but epithelial cell differentiation markers were distributed similarly in both strains. There was no evidence of excess extracellular matrix deposition or inflammation at the time points examined. In contrast, by adulthood MMP‐2−/− mice had normal alveolar size and structure, indicating normal alveolar development was not dependent on the ability of MT1‐MMP to activate pro–MMP‐2. These data indicate that MT1‐MMP is required for normal lung development. Developmental Dynamics 232:1079–1090, 2005.

Complementary Roles of Intracellular and Pericellular Collagen Degradation Pathways In Vivo

ABSTRACT Collagen degradation is essential for cell migration, proliferation, and differentiation. Two key turnover pathways have been described for collagen: intracellular cathepsin-mediated degradation and pericellular collagenase-mediated degradation. However, the functional relationship between these two pathways is unclear and even controversial. Here we show that intracellular and pericellular collagen turnover pathways have complementary roles in vivo. Individual deficits in intracellular collagen degradation (urokinase plasminogen activator receptor-associated protein/Endo180 ablation) or pericellular collagen degradation (membrane type 1-matrix metalloproteinase ablation) were compatible with development and survival. Their combined deficits, however, synergized to cause postnatal death by severely impairing bone formation. Interestingly, this was mechanistically linked to the proliferative failure and poor survival of cartilage- and bone-forming cells within their collagen-rich microenvironment. These findings have important implications for the use of pharmacological inhibitors of collagenase activity to prevent connective tissue destruction in a variety of diseases.