Lars H. Engelholm

A urokinase receptor-associated protein with specific collagen binding properties.

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 receptor-associated 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.

Extracellular Collagenases and the Endocytic Receptor, Urokinase Plasminogen Activator Receptor-associated Protein/Endo180, Cooperate in Fibroblast-mediated Collagen Degradation

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, fibroblast-mediated 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.

The non-phagocytic route of collagen uptake: a distinct degradation pathway.

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.

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.

Urokinase receptor-associated protein (uPARAP) is expressed in connection with malignant as well as benign lesions of the human breast and occurs in specific populations of stromal cells.

The urokinase‐type plasminogen activator (uPA) and the uPA receptor (uPAR) are key components in the plasminogen activation system, serving to promote specific events of extracellular matrix degradation in connection with tissue remodeling and cancer invasion. We recently described a new uPAR‐associated protein (uPARAP), an internalization receptor that interacts with the pro‐uPA:uPAR complex. In our study, we generated a specific polyclonal peptide antibody against human uPARAP and used it for the localization of uPARAP in different breast lesions. The affinity‐purified antibodies specifically recognized uPARAP in Western blotting and gave a strong signal in immunohistochemistry. The immunohistochemic localization pattern was found to be identical to that of uPARAP mRNA as determined in parallel by in situ hybridization. uPARAP expression was then studied in both benign and malignant breast lesions. Whereas the normal breast tissue was uPARAP‐negative, all benign lesions and ductal carcinoma in situ lesions showed immunoreactivity in fibroblast‐like cells and myoepithelial cells associated with the lesion. In invasive carcinoma, uPARAP immunoreactivity was limited to tumor‐associated mesenchymal cells. Double immunofluorescence analysis of invasive ductal carcinoma using antibodies against specific cell markers showed that uPARAP was localized in myofibroblasts and macrophages. No malignant cells, no endothelial cells and no vascular smooth muscle cells showed uPARAP immunoreactivity. We conclude that expression of uPARAP is associated with the abnormal breast and that expression appears in myofibroblasts, macrophages and myoepithelium. We suggest that uPARAP is involved in the clearance of the uPA:uPAR complex as well as other possible ligands during benign and malignant tissue remodeling.

Increased Expression of the Collagen Internalization Receptor uPARAP/Endo180 in the Stroma of Head and Neck Cancer

Local growth, invasion, and metastasis of malignancies of the head and neck involve extensive degradation and remodeling of the underlying, collagen-rich connective tissue. Urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 is an endocytic receptor recently shown to play a critical role in the uptake and intracellular degradation of collagen by mesenchymal cells. As a step toward determining the putative function of uPARAP/Endo180 in head and neck cancer progression, we used immunohistochemistry to determine the expression of this collagen internalization receptor in 112 human squamous cell carcinomas and 19 normal or tumor-adjacent head and neck tissue samples from the tongue, gingiva, cheek, tonsils, palate, floor of mouth, larynx, maxillary sinus, upper jaw, nasopharynx/nasal cavity, and lymph nodes. Specificity of detection was verified by staining of serial sections with two different monoclonal antibodies against two non-overlapping epitopes on uPARAP/Endo180 and by the use of isotype-matched non-immune antibodies. uPARAP/Endo180 expression was observed in stromal fibroblast-like, vimentin-positive cells. Furthermore, expression of the collagen internalization receptor was increased in tumor stroma compared with tumor-adjacent connective tissue or normal submucosal connective tissue and was most prominent in poorly differentiated tumors. These data suggest that uPARAP/Endo180 participates in the connective tissue destruction during head and neck squamous cell carcinoma progression by mediating cellular uptake and lysosomal degradation of collagen.

The collagen receptor uPARAP/Endo180.

The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem to operate in parallel. On certain cell types, uPARAP/Endo180 occurs in a complex with the urokinase plasminogen activator receptor (uPAR) where it seems to fulfill other functions in addition to collagenolysis. uPARAP/Endo180 is expressed on various mesenchymal cells, including subpopulations of fibroblasts, osteoblasts and chondrocytes, generally in conjunction with matrix turnover and collagenolysis. A striking expression is found in developing bone where the collagenolytic function of uPARAP/Endo180 is one of the rate-limiting steps in growth. In murine breast tumors, the endocytic function of the receptor in collagen breakdown seems to be involved in invasive tumor growth.

Tumor MMP-1 Activates Endothelial PAR1 to Facilitate Vascular Intravasation and Metastatic Dissemination

Intravasation, the active entry of primary tumor cells into the vasculature, remains the least studied step in the metastatic cascade. Protease-mediated escape and stromal invasion of tumor cells represent widely accepted processes leading up to the intravasation step. However, molecular factors that contribute directly to tumor cell vascular penetration have not been identified. In this study, the in vivo role of the collagenolytic protease, MMP-1, in cancer cell intravasation and metastasis was analyzed by using a highly disseminating variant of human HEp3 epidermoid carcinoma, HEp3-hi/diss. Although naturally acquired or experimentally induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation, supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors restored their impaired vascular dissemination. Surprisingly, abrogation of MMP-1 production and activity did not significantly affect HEp3-hi/diss migration or matrix invasion, suggesting noncollagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such noncollagenolytic mechanisms, MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly, MMP-1 deficiency led to decreased levels of intratumoral vascular permeability, tumor cell intravasation, and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells, we further show that endothelial PAR1 is a putative nontumor-cell/nonmatrix target, activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together, our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells.

The Urokinase Receptor Associated Protein (uPARAP/Endo180): A Novel Internalization Receptor Connected to the Plasminogen Activation System

The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While the biological consequences of this reaction have not yet been verified experimentally, a likely event is ligand internalization because uPARAP is a constitutively recycling internalization receptor. uPARAP also binds at least one component, collagen type V, in the extracellular matrix meshwork, pointing to a potential role in proteolytic substrate presentation. Additional ligands have been proposed, including collagenase-3 and glycoproteins capable of interacting with one of the multiple carbohydrate recognition-type domains of uPARAP. In various adult tissues uPARAP is present on fibroblasts, macrophages and a subset of endothelial cells. In fetal tissues the protein has also been demonstrated in certain bone forming regions. Hypotheses on the physiological function of uPARAP include regulatory roles in extracellular proteolysis. This type of function would be likely to direct the local turnover of proteases and their substrate degradation products and thus may add to the complicated interplay between several cell types in governing restricted tissue degradation.

Conformational Regulation of Urokinase Receptor Function IMPACT OF RECEPTOR OCCUPANCY AND EPITOPE-MAPPED MONOCLONAL ANTIBODIES ON LAMELLIPODIA INDUCTION

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the β-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the β-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.