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 ß1-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. ß1-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.

A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor.

The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.

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.

uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV.

Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient fibroblasts. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen IV in lysosomes in wild-type cells, but only very weak intracellular fluorescence accumulation in uPARAP/endo180-deficient fibroblasts. We conclude that uPARAP/endo180 is critical for targeted delivery of collagen IV to lysosomes for degradation implicating the receptor in normal and malignant extracellular matrix degradation. A similar localization pattern was observed for collagen V, suggesting that uPARAP/endo180 might be generally involved in collagen degradation.

uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion.

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.

Activated Galphaq family members induce Rho GTPase activation and Rho-dependent actin filament assembly.

Rho GTPase is required for actin filament assembly and serum response element (SRE)-dependent gene transcription. Certain G protein-coupled receptors (GPCRs) induce Rho-dependent responses, but the intermediary signaling steps are poorly understood. The heterotrimeric Galpha12 family can induce Rho-dependent responses. In contrast, there are conflicting reports on the role of the Galphaq family in Rho signaling. We report that expression of activated Galphaq members, or activation of endogenous Galphaq via GPCR stimulation, induces SRE reporter activation via Rho, and increased GTP-Rho levels. Moreover, microinjection of activated Galphaq in fibroblasts induces actin stress fiber formation via Rho. Galphaq functionally cooperates with Lbc Rho guanine nucleotide exchange factor. Overall, these findings indicate that Galphaq family signals are sufficient to induce Rho-dependent cellular responses.

The urokinase plasminogen activator receptor in the regulation of the actin cytoskeleton and cell motility.

Cell migration is a complex process requiring tight control of several mechanisms including dynamic reorganization of the actin cytoskeleton and adhesion to the extracellular matrix. The GPI-anchored urokinase plasminogen activator receptor (uPAR) has an important role in the regulation of cell motility in many cell types. This is partly due to the localization of proteolytic activity on the cell surface by binding of the serine protease uPA. Results accumulated over the last decade suggest that uPAR is also involved in motility control through other mechanisms. These include induction of signal transduction events after ligation with uPA, binding to the extracellular matrix molecule vitronectin (VN), and association with integrins and other transmembrane partners. In this review these mechanisms will be discussed with a special emphasis on how the GPI-linked receptor transmits signals to the intracellular milieu and how uPAR participates in the regulation of actin cytoskeleton reorganization and cell adhesion during cell migration.