The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N‐terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280‐289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C‐terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross‐linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

Matriptase/MT-SP1 is a novel tumor-associated type II transmembrane serine protease that is highly expressed in the epidermis, thymic stroma, and other epithelia. A null mutation was introduced into the Matriptase/MT-SP1 gene of mice to determine the role of Matriptase/ MT-SP1 in epidermal development and neoplasia. Matriptase/MT-SP1-de®cient mice developed to term but uniformly died within 48 h of birth. All epidermal surfaces of newborn mice were grossly abnormal with a dry, red, shiny, and wrinkled appearance. Matriptase/ MT-SP1-de®ciency caused striking malformations of the stratum corneum, characterized by dysmorphic and pleomorphic corneocytes and the absence of vesicular bodies in transitional layer cells. This aberrant skin development seriously compromised both inward and outward epidermal barrier function, leading to the rapid and fatal dehydration of Matriptase/MT-SP1-de®cient pups. Loss of Matriptase/MT-SP1 also seriously aected hair follicle development resulting in generalized follicular hypoplasia, absence of erupted vibrissae, lack of vibrissal hair canal formation, ingrown vibrissae, and wholesale abortion of vibrissal follicles. Furthermore, Matriptase/MT-SP1-de®ciency resulted in dramatically increased thymocyte apoptosis, and depletion of thymocytes. This study demonstrates that Matriptase/MT-SP1 has pleiotropic functions in the development of the epidermis, hair follicles, and cellular immune system.

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

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor–associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions.

Abstract. Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serumcontaining medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation.

SummaryBreakdowno ft he extracellular matrix is crucial forc ancer invasion and metastasis.Iti sa ccomplishedb yt he concerted action of several proteases, including the serine protease plasmin and anumberofmatrix metalloproteases.Theactivity of each of these proteasesisregulated by an arrayofactivators, inhibitors and cellular receptors.Thus,the generation of plasmininvolves the pro-enzyme plasminogen,the urokinaset ype plasminogen activator uPAa nd its pro-enzyme pro-uPA, the uPAi nhibitor PAI-1, the cell surfaceu PA receptor uPAR, and thep lasmin inhibitor α 2 -antiplasmin. Furthermore, the regulation of extracellular proteolysis in cancerinvolves acomplex interplaybetween cancercells and non-malignant stromalcells in the expression of themolecular components involved.For some types of cancer, this cellular interplaymimicsthat observedinthe tissueofori- KeywordsCancer invasion, plasminogen activation, matrix degradation, tissueremodelling, cancertherapy gin duringnon-neoplastic tissue remodellingprocesses.We proposethat cancer invasion can be considered as uncontrolled tissue remodelling. Inhibitiono fe xtracellular proteases is an attractivea pproach to cancer therapy.B ecause proteasesh ave manyd ifferentf unctions in the normal organism, efficienti nhibitionwill have toxic sideeffects.In cancerinvasion,like in normal tissue remodellingp rocesses, therea ppearst ob eafunctional overlapbetween differentextracellular proteases.This redundancymeans thatcombinationsofproteaseinhibitorsmust be used.Such combination therapy, however, is alsolikelytoincrease toxicity.Therefore foreach typeofcancer,acombination of proteasei nhibitorst hat is optimised with respectt ob oth maximal therapeutic effect and minimal toxic side effects need to be identified.

The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high molecular weight urokinase receptorassociated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices.

The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblastmediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis of large collagen fragments. First, we show that collagen that has been pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native collagen by uPARAP/Endo180-positive cells. Second, we demonstrate that this preference is governed by the acquisition of a gelatin-like structure by the collagen, occurring upon collagenase-mediated cleavage under native conditions. Third, we demonstrate that the growth of uPARAP/Endo180-deficient fibroblasts on a native collagen matrix leads to substantial extracellular accumulation of well defined collagen fragments, whereas, wild-type fibroblasts possess the ability to direct an organized and complete degradation sequence comprising both the initial cleavage, the endocytic uptake, and the intracellular breakdown of collagen.Collagens are the most abundant protein constituents of the extracellular matrix. The sheet-like collagens of the basement membrane and the fibrillar matrix collagens all incorporate into dense, insoluble protein networks that form a critical barrier against processes of cell migration such as those connected to tissue remodeling, including the invasive growth of cancer. Consequently, the degradation of these matrices is one of the rate-limiting steps in cancer invasion (1).The physiological mechanisms responsible for collagen degradation have long been subject to investigation. Due to their unique structural features, collagens can only be degraded by a minority of mammalian extracellular proteases, but certain matrix metalloproteases (MMPs), 3 such as MMP-1, MMP-2, MMP-8, MMP-13, and the membrane-bound MMP-14 and -15, are indeed active against native collagens (2-10). The initial attack of these proteases leads to the generation of well defined collagen fragments, which, while still in the extracellular environment, may be subject to further degradation by gelatinases, MMP-2 or MMP-9, or other types of proteases (11-13).Importantly, however, collagen may also be degraded through an intracellular turnover pathway (11,14). Recent studies have shown that an endocytic route of collagen breakdown, mediated by the collagen internalization recep...