Report |
Address correspondence to Thomas H. Bugge, Proteases and Tissue Remodeling Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Room 211, Bethesda, MD 20892. Tel.: (301) 435-1840. Fax: (301) 402-0823. E-mail: thomas.bugge{at}nih.gov
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Abstract |
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Key Words: cell adhesion; integrin; matrix internalization; matrix metalloproteinase; uPAR
* Abbreviations used in this paper: FN-II, fibronectin type II; MMP, matrix metalloproteinase; uPARAP, urokinase plasminogen activator receptorassociated protein.
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Introduction |
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The urokinase plasminogen activator receptorassociated protein (uPARAP)*/Endo180 is a novel multi-domain transmembrane glycoprotein that was identified through its specific interaction with receptor-bound pro-urokinase plasminogen activator on the surface of certain cultured cells (Behrendt et al., 2000) and, independently, as a constitutive endocytic recycling glycoprotein (Endo180) that is capable of internalizing mAbs directed against it (Howard and Isacke, 2002; Sheikh et al., 2000). uPARAP/Endo180 is a member of the macrophage mannose receptor family of type I transmembrane glycoproteins (Engelholm et al., 2001a). The distinct and highly conserved domain structure that characterizes this protein family includes an NH2-terminal, cysteine-rich ricin B lectin-like domain, a fibronectin type II (FN-II) domain, a series of 810 domains related to C-type carbohydrate recognition domains, a transmembrane domain, and a short COOH-terminal cytoplasmic tail (Sonnhammer et al., 1998; Bateman et al., 1999; Engelholm et al., 2001a). uPARAP/Endo180 is highly expressed on osteoblasts and osteocytes at sites of endochondral and intramembranous ossification during development (Engelholm et al., 2001b). The postnatal expression of uPARAP/Endo180 is restricted to specific subsets of fibroblasts, macrophages, and endothelial cells (Sheikh et al., 2000). The biochemical functions of uPARAP/Endo180 have yet to be elucidated. However, several conspicuous properties of the receptor suggest that uPARAP/Endo180 may be involved in the remodeling of the ECM and/or modulation of the localization or availability of soluble ligands in the pericellular environment (Hanasaki and Arita, 1999; Martinez-Pomares et al., 1999; Sheikh et al., 2000; Engelholm et al., 2001a). Of particular interest, initial studies on uPARAP/Endo180 revealed that the uPAR-dependent binding of pro-urokinase plasminogen activator to this receptor is blocked efficiently by low concentrations of collagen type V, suggesting a direct interaction with this constituent of the ECM. This function is likely to be mediated by the FN-II domain because this domain type is typically associated with collagen binding (Ancian et al., 1995). In this work, we generated a targeted deletion in the uPARAP/Endo180 gene and show that uPARAP/Endo180 is essential for the uptake of collagen by fibroblasts, and that it has important functions in fibroblast adhesion and migration on fibrillar collagen matrices.
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Results and discussion |
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Strikingly, the uPARAP/Endo180-deficient fibroblasts displayed a virtually complete abrogation of the cellular uptake of collagen types I, IV, and V (Fig. 2 A, panels 3, 4, and 1, respectively). This deficiency in collagen internalization was observed with several independent isolates of uPARAP/Endo180-/- fibroblasts and could be alleviated by the reintroduction of uPARAP/Endo180 by transient transfection of the cells with a uPARAP/Endo180 cDNA expression vector (Fig. 2 A, panel 2; unpublished data). In contrast, the cellular uptake of holotransferrin, MMP-13, and fibronectin was unaffected (Fig. 2 A, panels 57). The internalization of MMP-13 has been shown previously to be uPARAP/Endo180-independent (Bailey et al., 2002), but depends on the low density lipoprotein receptorrelated protein (LRP; Barmina et al., 1999). Therefore, as an additional test of our assay system, we included samples with LRP-deficient cells (LRP-/- and matched controls) in the same manner to study the internalization of 125I-labeled MMP-13. This experiment confirmed the essential role of LRP in this process (P < 0.0007), whereas LRP deficiency had no effect on the cellular uptake of 125I-labeled collagen, examined as described for uPARAP/Endo180-/- cells (unpublished data). These data show that uPARAP/Endo180 has a critical and specific role in the uptake of collagen by fibroblasts.
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The cellular uptake of collagen has been reported to be insensitive to inhibitors of MMPs, serine proteases, and cysteine proteases, and extensive degradation of collagen before the internalization step does not seem to be needed (Everts et al., 1988, 1989). The major degradation processes in this pathway occur after the cellular uptake event, by the fusion of collagen-containing intracellular vesicles with lysosomes and the degradation of acid-denatured collagen by lysosomal proteases (Everts et al., 1988, 1996; Everts and Beertsen, 1992). Our current demonstration of a crucial function of uPARAP/Endo180 in the actual cellular internalization of collagen thus addresses a central step in this series of events.
uPARAP/Endo180-deficient cells display delayed adhesion to collagen matrices
We used a standard cell adhesion assay, in which cells are allowed to attach briefly to immobilized collagens, to investigate if the lack of uPARAP/Endo180 could directly influence cell adhesion. Loss of uPARAP/Endo180 resulted in a 50% reduction in the adhesion of fibroblasts to type V collagen-coated surfaces (Fig. 3 D). This impairment was observed in comparisons of several independent isolates of uPARAP/Endo180-/- fibroblasts with littermate control uPARAP/Endo180+/+ fibroblasts (unpublished data). Interestingly, uPARAP/Endo180-/- cells also demonstrated a reduced ability to adhere to other immobilized collagens, including purified types I and IV collagen and total tendon collagen (Fig. 3, A, C, and E), although the reduction in these cases was less dramatic. In contrast, uPARAP/Endo180 deficiency did not affect the adhesion of fibroblasts to a noncollagen substrate such as fibronectin (Fig. S2 C) or a surface coated with total serum proteins (Fig. 3 F). A time-course study of cell adhesion to collagen V and collagen I showed that the effect of uPARAP/Endo180 deficiency was limited to the initial phase (1530 min after plating), and wild-type and targeted cells reached the same level of adhesion after 12 h (unpublished data).
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These experiments revealed that uPARAP/Endo180 has an early modulatory function in fibroblast adhesion to collagen matrices, whereas ß1 integrins appear to be indispensable for this process. Thus, our observations make it tempting to speculate that uPARAP/Endo180 is critical for fully effective initial cellular interactions with collagen, this role having an impact on collagen adhesion, beyond its crucial role in collagen internalization. A simple cell binding assay with solubilized 125I-labeled collagen V at 4°C supported this notion, as a >50% reduction in binding was observed with uPARAP/Endo180-/- cells (unpublished data).
uPARAP/Endo180 deficiency impairs the migration of fibroblasts on collagen fibrils
Cell migration is intimately linked with adhesion to the ECM (Murphy and Gavrilovic, 1999). To directly test if uPARAP is important for cellular migration on collagen, we performed single-cell, parallel time-lapse video microscopy of matched pairs of primary dermal uPARAP/Endo180-/- and littermate control fibroblasts on a mixed fibrillar collagen matrix (Fig. 4, A and B). uPARAP/Endo180-/- cells demonstrated a significant and reproducible impairment in their migration, displaying a >30% reduction in the average migration rate. The mechanistic details of this effect await further studies. One role of uPARAP/Endo180 in cell migration may be directly related to increasing cellular adhesion. However, uPARAP/Endo180-mediated collagen internalization may also promote migration by increasing adhesion site turnover, thereby facilitating cell spreading. Finally, the interplay between uPARAP/Endo180 and integrins may influence integrin-mediated signaling.
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Materials and methods |
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Western blot analysis
Synthesis of peptide 809829 of the murine uPARAP/Endo180 sequence (GenBank/EMBL/DDBJ accession no. AAC52729), immunization of rabbits, affinity purification of antibodies and Western blotting of cell lysates were performed as described previously (Schnack Nielsen et al., 2002) using the antibody at a final concentration of 2 µg/ml.
Ligand internalization assay
Primary skin fibroblasts were isolated from neonates as described previously (Holmbeck et al., 1999). 20 µg type I collagen, type IV collagen, type V collagen, MMP-13, human holotransferrin (all from Calbiochem-Novachem), and human plasma fibronectin (Sigma-Aldrich) were labeled with 125I as described previously (Behrendt et al., 1996). Cellular ligand internalization assays were performed as described previously (Hahn-Dantona et al., 2001). In brief, samples of 105 cells were seeded in 24-well tissue culture plates and cultured in DME, 10% FCS until near confluence. The cells were washed gently in DME at 37°C and were then cultured for at least 1 h in binding buffer (DME, 20 mM Hepes, pH 7.4, 15 mg/ml BSA, and 1x Nutridoma-[SP] serum substitute; Roche). The medium was removed and replaced by binding buffer with 125I-labeled protein ranging in concentration from 0.5 to 5 nM, followed by incubation at 37°C. In some experiments, the cells were preincubated for 30 min at 4°C with 10 µg/ml anti-ß1 antibody (HA2/5) or 10 µg/ml isotype-matched control anti-TNP antibody (G2351; both from BD Biosciences) before the addition of labeled ligand. The cells were then washed twice with ice-cold PBS, and incubated <2 min at 4°C with 50 µg/ml trypsin, 50 µg/ml proteinase K, and 0.53 mM EDTA in HBSS. The detached cells were centrifuged at 3,000 rpm for 5 min at 4°C, and the radioactivity in the pellet (internalized material) and supernatant (surface-released material) was measured in a gamma counter. Statistical significance was calculated by two-tailed t test.
Transient transfection of primary fibroblasts
Transfection of cultured fibroblasts was performed essentially as described previously (Kjøller and Hall, 2001), using the vector pcDNA3-uPARAP/Endo180 (provided by Dr. Clare Isacke, Chester Beatty Laboratories, London, UK; Sheikh et al., 2000). The transfection efficiency, estimated by determination of the percentage of GFP-expressing cells after cotransfection with pEGF-P1 (CLONTECH Laboratories, Inc.) was 10%.
Cell adhesion assay
96-well plates were coated for 1 h at 37°C with either 10% FCS in DME, or with rat tail tendon collagen (provided by Dr. Jack Windsor, University of Indianapolis, Indianapolis, IN), human type I collagen, murine type II collagen, human type IV collagen, or human type V collagen (all from Calbiochem-Novabiochem">Calbiochem-Novabiochem), at 20 µg/ml in 10 mM acetic acid. The coated wells were washed three times with PBS, and the residual binding sites were blocked by incubation with 0.2% BSA in PBS for 1 h at 37°C, followed by three additional washes with PBS at RT. Primary fibroblasts were detached by mild trypsinization, washed, resuspended in serum-free medium with 0.2% BSA, and incubated at 37°C for 30 min with gentle rotation to recover from trypsinization. Cell viability was determined by Trypan blue exclusion. 5 x 104 cells were added to each well and were allowed to attach for 30 min at 37°C. Nonadherent cells were removed by two gentle washes with 200 µl PBS, and the number of adherent cells was determined by MTT analysis (Liu et al., 2001). In some experiments, cells were preincubated with anti-ß1 antibody or control antibody as specified above. Statistical significance was calculated by two-tailed t test.
Cell migration assay
Tissue culture plates with a glass coverslip bottom (MatTek Corporation) were coated with rat tail tendon collagen as described above. The dishes were washed twice with PBS, and incubated for 1 h at 37°C with 2% heat-denatured BSA in PBS to block residual binding sites. The integrity of the collagen fibrils was verified by incubation with purified trypsin and MMP-9 (Netzel-Arnett et al., 2002). uPARAP/Endo180-/- or uPARAP/Endo180-expressing (uPARAP/Endo180+/+ or +/-) littermate control fibroblasts (1,000 cells/well) were seeded overnight on the collagen layer in DME containing 10% FCS. FCS was included during seeding and microscopy due to the long duration of the experiments. Cell migration was analyzed by time-lapse video microscopy as described previously (Gu et al., 1999). In brief, cell movements were recorded using inverted microscopes (Carl Zeiss MicroImaging, Inc.), collecting video images with tube cameras (Newvicon model 2400; Hamamatsu Photonics) at 10-min intervals for at least 6 h. From the individual cell tracks, cell velocities were calculated using MetaMorph® 4.6 software (Universal Imaging Corporation). A one-way ANOVA and a Bonferroni multiple comparisons post-test were performed using InStat® software (GraphPad Software, Inc.) to determine the statistical significance between samples. The experiment was repeated with four independently isolated pairs of uPARAP/Endo180-/- and littermate-matched uPARAP/Endo180+ fibroblasts, and included a total of 52 and 82 individual measurements, respectively.
Flow cytometry
Cells were detached in PBS with 5 mM EDTA, 5 mg/ml BSA, washed twice in binding buffer (PBS with 0.1 mg/ml CaCl2, 0.05 mg/ml MgCl2, 1 mg/ml NaN3, and 5 mg/ml BSA), and incubated for 30 min at 4°C with either FITC-conjugated anti-ß1 integrin antibody (HA2/5) or FITC-conjugated isotype-matched control anti-TNP antibody (G2351), using 5 µg of antibody for 106 cells in 200 µl binding buffer. After incubation, cells were washed twice in ice-cold binding buffer and fixed in 1% PFA in binding buffer without NaN3. Flow cytometry analysis was then performed using a FACsortTM instrument (Becton Dickinson).
Online supplemental material
Supplemental materials and methods, and additional primary data are available at (http://www.jcb.org/cgi/content/full/jcb.200211091/DC1).
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Acknowledgments |
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This work was supported by grants from the Danish Cancer Society, the Danish Cancer Research Foundation, the Danish Research Council, and the Danish Biotechnology Program (to L.H. Engelholm, K. Dano, and N. Behrendt).
Submitted: 20 November 2002
Revised: 13 February 2003
Accepted: 14 February 2003
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