Portal de Periódicos da CAPES

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

2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês

10.1074/jbc.m701088200

1083-351X

Daniel H. Madsen, Lars H. Engelholm, Signe Ingvarsen, Thore Hillig, Rebecca A. Wagenaar-Miller, Lars Kjøller, Henrik Gårdsvoll, Gunilla Høyer‐Hansen, Kenn Holmbeck, Thomas Bugge, Niels Behrendt,

Cell Adhesion Molecules Research

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 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. 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 (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5244) Google Scholar). 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), 3The abbreviations used are:MMPmatrix metalloproteaseuPARAPurokinase plasminogen activator receptor-associated protein (also designated Endo180)suPARAPsoluble uPARAP fusion proteinFITCfluorescein isothiocyanateAPMA4-aminophenylmercuric acetate such as MMP-1, MMP-2, MMP-8, MMP-13, and the membrane-bound MMP-14 and -15, are indeed active against native collagens (2Stricklin G.P. Bauer E.A. Jeffrey J.J. Eisen A.Z. Biochemistry. 1977; 16: 1607-1615Crossref PubMed Scopus (277) Google Scholar, 3Aimes R.T. Quigley J.P. J. Biol. Chem. 1995; 270: 5872-5876Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 4Freije J.M. ez-Itza I. Balbin M. Sanchez L.M. Blasco R. Tolivia J. Lopez-Otin C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar, 5Knauper V. Lopez-Otin C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 6Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar, 7Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar, 8Hotary K. Allen E. Punturieri A. Yana I. Weiss S.J. J. Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (514) Google Scholar, 9Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (301) Google Scholar, 10Netzel-Arnett S. Mitola D.J. Yamada S.S. Chrysovergis K. Holmbeck K. Birkedal-Hansen H. Bugge T.H. J. Biol. Chem. 2002; 277: 45154-45161Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 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 (11Mohamed M.M. Sloane B.F. Nat. Rev. Cancer. 2006; 6: 764-775Crossref PubMed Scopus (1046) Google Scholar, 12Murphy G. Reynolds J.J. Bretz U. Baggiolini M. Biochem. J. 1982; 203: 209-221Crossref PubMed Scopus (169) Google Scholar, 13Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar). matrix metalloprotease urokinase plasminogen activator receptor-associated protein (also designated Endo180) soluble uPARAP fusion protein fluorescein isothiocyanate 4-aminophenylmercuric acetate Importantly, however, collagen may also be degraded through an intracellular turnover pathway (11Mohamed M.M. Sloane B.F. Nat. Rev. Cancer. 2006; 6: 764-775Crossref PubMed Scopus (1046) Google Scholar, 14Arora P.D. Manolson M.F. Downey G.P. Sodek J. McCulloch C.A. J. Biol. Chem. 2000; 275: 35432-35441Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Recent studies have shown that an endocytic route of collagen breakdown, mediated by the collagen internalization receptor, uPARAP/Endo180 (15Behrendt N. Jensen O.N. Engelholm L.H. Mortz E. Mann M. Dano K. J. Biol. Chem. 2000; 275: 1993-2002Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Sheikh H. Yarwood H. Ashworth A. Isacke C.M. J. Cell Sci. 2000; 113 (Pt 6): 1021-1032Crossref PubMed Google Scholar, 17Engelholm L.H. List K. Netzel-Arnett S. Cukierman E. Mitola D.J. Aaronson H. Kjoller L. Larsen J.K. Yamada K.M. Strickland D.K. Holmbeck K. Dano K. Birkedal-Hansen H. Behrendt N. Bugge T.H. J. Cell Biol. 2003; 160: 1009-1015Crossref PubMed Scopus (146) Google Scholar) (hereafter designated uPARAP), is a rate-limiting factor in collagenolysis and invasive growth of breast tumors in mice (18Curino A.C. Engelholm L.H. Yamada S.S. Holmbeck K. Lund L.R. Molinolo A.A. Behrendt N. Nielsen B.S. Bugge T.H. J. Cell Biol. 2005; 169: 977-985Crossref PubMed Scopus (115) Google Scholar). This new observation underscores the importance of understanding the function of uPARAP and the interplay between cellular and extracellular events in collagen and matrix turnover. uPARAP is a type-1 transmembrane protein and a member of the mannose receptor protein family of constitutive endocytosis receptors (reviewed in Refs. 19East L. Isacke C.M. Biochim. Biophys. Acta. 2002; 1572: 364-386Crossref PubMed Scopus (515) Google Scholar and 20Behrendt N. Biol. Chem. 2004; 385: 103-136Crossref PubMed Scopus (86) Google Scholar). The extracellular portion includes a cysteine-rich domain, a fibronectin type-II domain, and eight consecutive C-type lectin-like domains (20Behrendt N. Biol. Chem. 2004; 385: 103-136Crossref PubMed Scopus (86) Google Scholar). The receptor binds directly to various types of collagen through interactions governed, wholly or in part, by the fibronectin type-II domain (15Behrendt N. Jensen O.N. Engelholm L.H. Mortz E. Mann M. Dano K. J. Biol. Chem. 2000; 275: 1993-2002Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 21East L. McCarthy A. Wienke D. Sturge J. Ashworth A. Isacke C.M. EMBO Rep. 2003; 4: 710-716Crossref PubMed Scopus (87) Google Scholar, 22Thomas E.K. Nakamura M. Wienke D. Isacke C.M. Pozzi A. Liang P. J. Biol. Chem. 2005; 280: 22596-22605Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 23Wienke D. MacFadyen J.R. Isacke C.M. Mol. Biol. Cell. 2003; 14: 3592-3604Crossref PubMed Scopus (124) Google Scholar). uPARAP also has carbohydrate binding activity, mediated through the second C-type lectin-like domain (24East L. Rushton S. Taylor M.E. Isacke C.M. J. Biol. Chem. 2002; 277: 50469-50475Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and on certain cell types it interacts with the urokinase plasminogen activator receptor (15Behrendt N. Jensen O.N. Engelholm L.H. Mortz E. Mann M. Dano K. J. Biol. Chem. 2000; 275: 1993-2002Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 25Behrendt N. Ronne E. Dano K. FEBS Lett. 1993; 336: 394-396Crossref PubMed Scopus (22) Google Scholar) in a process implicated in signal transduction in connection with cell migration (26Sturge J. Wienke D. East L. Jones G.E. Isacke C.M. J. Cell Biol. 2003; 162: 789-794Crossref PubMed Scopus (65) Google Scholar, 27Sturge J. Wienke D. Isacke C.M. J. Cell Biol. 2006; 175: 337-347Crossref PubMed Scopus (61) Google Scholar). The molecular interaction between uPARAP and collagen is reflected in part in a decreased initial adhesion of uPARAP-deficient cells on a collagen matrix, but a much more striking effect is noted on the ability of fibroblasts to internalize solubilized collagen, which is lost completely upon gene targeting of uPARAP (17Engelholm L.H. List K. Netzel-Arnett S. Cukierman E. Mitola D.J. Aaronson H. Kjoller L. Larsen J.K. Yamada K.M. Strickland D.K. Holmbeck K. Dano K. Birkedal-Hansen H. Behrendt N. Bugge T.H. J. Cell Biol. 2003; 160: 1009-1015Crossref PubMed Scopus (146) Google Scholar, 23Wienke D. MacFadyen J.R. Isacke C.M. Mol. Biol. Cell. 2003; 14: 3592-3604Crossref PubMed Scopus (124) Google Scholar, 28Kjoller L. Engelholm L.H. Hoyer-Hansen M. Dano K. Bugge T.H. Behrendt N. Exp. Cell Res. 2004; 293: 106-116Crossref PubMed Scopus (88) Google Scholar). uPARAP has also been shown to be responsible for the uptake of collagen in hepatic stellate cells (29Mousavi S.A. Sato M. Sporstol M. Smedsrod B. Berg T. Kojima N. Senoo H. Biochem. J. 2005; 387: 39-46Crossref PubMed Scopus (32) Google Scholar). Following endocytosis from clathrin-coated pits (30Isacke C.M. van der G.P. Hunter T. Trowbridge I.S. Mol. Cell. Biol. 1990; 10: 2606-2618Crossref PubMed Scopus (57) Google Scholar, 31Howard M.J. Isacke C.M. J. Biol. Chem. 2002; 277: 32320-32331Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), uPARAP is routed to early endosomes and later recycled to the cell surface, whereas, the initially bound collagen is routed to lysosomes for degradation (23Wienke D. MacFadyen J.R. Isacke C.M. Mol. Biol. Cell. 2003; 14: 3592-3604Crossref PubMed Scopus (124) Google Scholar, 28Kjoller L. Engelholm L.H. Hoyer-Hansen M. Dano K. Bugge T.H. Behrendt N. Exp. Cell Res. 2004; 293: 106-116Crossref PubMed Scopus (88) Google Scholar). The latter process is primarily undertaken by cysteine proteases (11Mohamed M.M. Sloane B.F. Nat. Rev. Cancer. 2006; 6: 764-775Crossref PubMed Scopus (1046) Google Scholar, 32Sameni M. Dosescu J. Sloane B.F. Biol. Chem. 2001; 382: 785-788Crossref PubMed Google Scholar). Consistent with a role in matrix turnover, the expression of uPARAP is clearly associated with sites of tissue remodeling. Thus, the receptor is expressed in osteoblasts of developing bone (33Engelholm L.H. Nielsen B.S. Netzel-Arnett S. Solberg H. Chen X.D. Lopez Garcia J.M. Lopez-Otin C. Young M.F. Birkedal-Hansen H. Dano K. Lund L.R. Behrendt N. Bugge T.H. Lab. Invest. 2001; 81: 1403-1414Crossref PubMed Scopus (63) Google Scholar) and in myofibroblasts and certain other cell types in wound healing tissue (34Honardoust H.A. Jiang G. Koivisto L. Wienke D. Isacke C.M. Larjava H. Hakkinen L. Histopathology. 2006; 49: 634-648Crossref PubMed Scopus (31) Google Scholar). In connection with cancer, expression is observed in the myoepithelium of mammary carcinoma in situ, in the myofibroblasts of invasive mammary carcinoma, and in fibroblast-like cells in oral cancer (35Schnack N.B. Rank F. Engelholm L.H. Holm A. Dano K. Behrendt N. Int. J. Cancer. 2002; 98: 656-664Crossref PubMed Scopus (56) Google Scholar, 36Sulek J. Wagenaar-Miller R.A. Shireman J. Molinolo A. Madsen D.H. Engelholm L.H. Behrendt N. Bugge T.H. J. Histochem. Cytochem. 2007; 55: 347-353Crossref PubMed Scopus (48) Google Scholar). Little is known, however, about the interplay between the intracellular and extracellular processes that govern the breakdown of extracellular matrix components. In this work, we show that fibroblasts, a dominant cell type in collagen turnover, direct an organized degradation pathway that combines extracellular cleavage and uPARAP-mediated endocytosis. These observations assign a new function to uPARAP as an efficient endocytic receptor for early collagen cleavage products. Proteins and Cultured Cells—Skin fibroblasts from newborn homozygous uPARAP-deficient mice and littermate control wild-type mice were isolated and cultured as published previously (17Engelholm L.H. List K. Netzel-Arnett S. Cukierman E. Mitola D.J. Aaronson H. Kjoller L. Larsen J.K. Yamada K.M. Strickland D.K. Holmbeck K. Dano K. Birkedal-Hansen H. Behrendt N. Bugge T.H. J. Cell Biol. 2003; 160: 1009-1015Crossref PubMed Scopus (146) Google Scholar). Skin fibroblasts from mice homozygously deficient for MMP-2 (37Itoh T. Ikeda T. Gomi H. Nakao S. Suzuki T. Itohara S. J. Biol. Chem. 1997; 272: 22389-22392Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), MMP-13 (38Stickens D. Behonick D.J. Ortega N. Heyer B. Hartenstein B. Yu Y. Fosang A.J. Schorpp-Kistner M. Angel P. Werb Z. Development. 2004; 131: 5883-5895Crossref PubMed Scopus (501) Google Scholar), or MMP-14 (7Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar) were isolated and cultured in the same manner, in each case accompanied by wild-type control cultures obtained from littermate control mice. Recombinant, soluble uPARAP fusion protein (suPARAP), comprising the extracellular portion (Met1–Ser1402) of the human uPARAP sequence fused to a purification tag (the third domain of the urokinase receptor), was expressed in Drosophila Schneider S2 cells and purified as described (39Gardsvoll H. Hansen L.V. Jorgensen T.J. Ploug M. Protein Expr. Purif. 2007; 52: 384-394Crossref PubMed Scopus (36) Google Scholar). The monoclonal mouse anti-uPARAP antibody 2.h.9:F12 was raised against purified suPARAP and produced as described previously (36Sulek J. Wagenaar-Miller R.A. Shireman J. Molinolo A. Madsen D.H. Engelholm L.H. Behrendt N. Bugge T.H. J. Histochem. Cytochem. 2007; 55: 347-353Crossref PubMed Scopus (48) Google Scholar). The following proteins were purchased from the commercial sources indicated: native trypsin-resistant collagen type-I from rat tail (BD Biosciences), FITC-collagen type I from MD Biosciences (Zürich, Switzerland), cysteine protease inhibitor E-64d and recombinant, human pro-MMP-13 (Calbiochem, San Diego, CA), 4-aminophenylmercuric acetate (APMA) (Sigma-Aldrich), interleukin 1β, and tumor necrosis factor-α (Peprotech, Rocky Hill, NJ), and polyclonal rabbit antibody against collagen type-I (Rockland, Gilbertsville, PA). Cleavage of Collagen under Non-denaturing Conditions—For activation of pro-MMP-13, the pro-enzyme was diluted to 50 μg/ml in reaction buffer (100 mm Tris-HCl, 10 mm CaCl2, 1 μm ZnCl2, 100 mm NaCl, pH 7.0). APMA was added from a freshly prepared 20 mm stock solution in 0.1 m NaOH to a final concentration of 1 mm, after which the sample was incubated for 30 min at 37 °C. For the removal of APMA after the activation step, the sample was subjected to four rounds of buffer exchange, each cycle including centrifugation through an Amicon Ultra-free-MC 10,000 NMWL Filter Unit, to 10% of the starting volume and replenishment of reaction buffer to 100% volume. For cleavage of collagen, collagen was diluted to 250 μg/ml in reaction buffer, after which the freshly activated MMP-13 was added to a final concentration of 8.75 μg/ml (∼175 nm). The samples were incubated at 25 °C for 24 h. Internalization Studies with Collagen and Collagen Fragments—Labeling of proteins with 125I and radioligand internalization assays with cultured newborn mouse fibroblasts were performed as described before (17Engelholm L.H. List K. Netzel-Arnett S. Cukierman E. Mitola D.J. Aaronson H. Kjoller L. Larsen J.K. Yamada K.M. Strickland D.K. Holmbeck K. Dano K. Birkedal-Hansen H. Behrendt N. Bugge T.H. J. Cell Biol. 2003; 160: 1009-1015Crossref PubMed Scopus (146) Google Scholar) and included incubation of cells with radioligand in 24-well tissue culture plates for 3 h at 37 °C, followed by isolation of the intracellular fraction and quantification of the internalized radioactivity. Before the assay, the protein concentration and the specific radioactivity of each of the radiolabeled protein preparations was estimated by SDS-PAGE and silver staining, by careful comparison with a narrow dilution series of a known standard of the same protein preparation in the non-labeled form, run on the same gel. 30 ng of labeled intact or cleaved collagen was added to each well with near-confluent cells in a volume of 300 μl. In some experiments, the labeled collagen ligands were preincubated at elevated temperature before the assay, as indicated. Incubation of cultured fibroblasts with FITC-labeled intact and cleaved collagen-I (15 μg/ml) followed by confocal fluorescence microscopy with a Zeiss LSM 510 microscope was performed as described previously for fluorescence-labeled, intact collagen-IV (28Kjoller L. Engelholm L.H. Hoyer-Hansen M. Dano K. Bugge T.H. Behrendt N. Exp. Cell Res. 2004; 293: 106-116Crossref PubMed Scopus (88) Google Scholar), using an incubation period of 22 h in the presence of 20 μm E-64d. Surface Plasmon Resonance Analysis—Interaction analyses with intact and cleaved collagen and immobilized suPARAP were done using a BIAcore2000 instrument (BIAcore, Uppsala, Sweden). Before interaction analysis, the monoclonal antibody 2.h.9:F12, against uPARAP (36Sulek J. Wagenaar-Miller R.A. Shireman J. Molinolo A. Madsen D.H. Engelholm L.H. Behrendt N. Bugge T.H. J. Histochem. Cytochem. 2007; 55: 347-353Crossref PubMed Scopus (48) Google Scholar), was coupled to the surface of a CM5-type BIAcore chip by amine-directed coupling, following the instructions provided by the manufacturer. This chip was then used as a catching support for suPARAP, injected subsequently, to obtain a level of immobilized suPARAP of ∼5500 resonance units. Due to the binding characteristics of the 2.h.9: F12 antibody, this non-covalent immobilization step was irreversible under the conditions used, because the elution conditions for collagen (below) led to no release of bound suPARAP from the chip. Cleaved or intact collagen type-I was preincubated in assay buffer (0.01 m Hepes, 0.15 m NaCl, 1 mm CaCl2, 0.005% surfactant P20, pH 7.4) at 37 °C, or at elevated temperature as indicated, after which various concentrations of the protein were injected. After each round of binding and dissociation, the chip was regenerated by injection of 10 mm glycine/HCl, pH 2.0, to obtain complete dissociation of all bound collagen material. A parallel flow channel, containing coupled antibody but no suPARAP, served as a control for unspecific binding and buffer bulk subtraction. No binding of intact or cleaved collagen was obtained with antibody alone. The BIAcore interaction analyses were otherwise performed as described (40Behrendt N. Ronne E. Dano K. J. Biol. Chem. 1996; 271: 22885-22894Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), monitoring binding and dissociation at 20 °C in assay buffer at a flow rate of 10 μl/min. Curve fitting to kinetic models and calculation of kinetic parameters were done using the BIAeval software, version 3.2 RC1, supplied with the instrument. Analysis of Collagen Turnover in Growing Fibroblasts—A stock solution of collagen type-I in 0.02 m acetic acid was brought to neutral pH by addition of the appropriate volume of phosphate buffer to obtain a final concentration of 300 μg/ml collagen, after which a trace amount of 125I-labeled collagen type-I was added to obtain a radioactivity of ∼1,500,000 cpm/ml. Immediately thereafter, each well of a 24-well tissue culture plate was coated with 350 μl of the tracer-containing collagen solution. The collagen was gelled by incubation at 37 °C for 3 h and allowed to dry in a flow hood. For rehydration, the dried gel was washed with three changes of water, after which the wells were incubated with phosphate-buffered saline at room temperature for 24 h. Phosphate-buffered saline was removed, and 1 × 105 cells were added to each well in a volume of 300 μl of Dulbecco's modified Eagle's medium with 10% fetal calf serum, covering the bottom of the well. The cells were allowed to attach for 4 h, after which the medium was removed and replaced with 400 μl of Dulbecco's modified Eagle's medium, including 1 nm interleukin 1β and 10 nm tumor necrosis factor-α. After 5 days of cell culture, the conditioned medium from each well was collected. The media were analyzed by SDS-PAGE followed by Western blotting or automated phosphorimaging analysis as described previously (17Engelholm L.H. List K. Netzel-Arnett S. Cukierman E. Mitola D.J. Aaronson H. Kjoller L. Larsen J.K. Yamada K.M. Strickland D.K. Holmbeck K. Dano K. Birkedal-Hansen H. Behrendt N. Bugge T.H. J. Cell Biol. 2003; 160: 1009-1015Crossref PubMed Scopus (146) Google Scholar). After harvest of the media, the cells and residual matrix were washed with phosphate-buffered saline. Cells were then detached by trypsinization, after which the matrix was stained with Coomassie Blue for visualization of zones of collagen clearance, as described before (7Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar). uPARAP-dependent Internalization and Degradation of Collagen Fragments—Because the extracellular routes of collagen turnover are initiated by a well defined cleavage in the triple helical structure (41Highberger J.H. Corbett C. Gross J. Biochem. Biophys. Res. Commun. 1979; 89: 202-208Crossref PubMed Scopus (23) Google Scholar), we wanted to learn whether the same initial event might serve as a starting point for the endocytic collagen degradation route in fibroblasts. Consequently, we tested whether defined collagen fragments, resulting from treatment of native collagen with a mammalian collagenase, can serve as ligands for uPARAP-mediated internalization. Trypsin-resistant rat tail collagen type-I was treated with recombinant collagenase-3 (MMP-13) at 25 °C to obtain complete cleavage into ¼ and ¾ fragments (Fig. 1A), in accordance with the pattern known for the cleavage of native collagen (42Messent A.J. Tuckwell D.S. Knauper V. Humphries M.J. Murphy G. Gavrilovic J. J. Cell Sci. 1998; 111 (Pt 8): 1127-1135Crossref PubMed Google Scholar, 43Chung L. Dinakarpandian D. Yoshida N. Lauer-Fields J.L. Fields G.B. Visse R. Nagase H. EMBO J. 2004; 23: 3020-3030Crossref PubMed Scopus (381) Google Scholar). For the subsequent internalization studies, the intact collagen and the collagen fragments were labeled with 125I (Fig. 1B). Analyses of uPARAP-mediated internalization were then undertaken, studying the endocytic activity of fibroblasts from newborn mice using radiolabeled collagen and collagen fragments as the ligands. This setup allowed studies on endocytosis in a system which, in the case of intact collagens, has been shown previously to be completely uPARAP-dependent (17Engelholm L.H. List K. Netzel-Arnett S. Cukierman E. Mitola D.J. Aaronson H. Kjoller L. Larsen J.K. Yamada K.M. Strickland D.K. Holmbeck K. Dano K. Birkedal-Hansen H. Behrendt N. Bugge T.H. J. Cell Biol. 2003; 160: 1009-1015Crossref PubMed Scopus (146) Google Scholar). Furthermore, the use of parallel cultures of fibroblasts from wild-type and littermate control uPARAP-deficient mice (uPARAP-/-) enabled us to also test this uPARAP dependence for unknown samples, such as the collagen fragments. This study revealed that both the intact and the cleaved collagen are taken up by wild-type (uPARAP+/+) cells (Fig. 2A, black columns). Strikingly, however, the uptake of the cleaved protein (column 5) was much more efficient than the internalization of the untreated collagen (column 1). This increased efficiency was indeed the result of proteolytic cleavage, because preincubation of intact collagen at 25 °C without collagenase-3 did not affect the internalization (column 3). The uptake of both cleaved and uncleaved collagen was completely uPARAP-dependent, because fibroblasts from littermate uPARAP-/- mice displayed no internalization (gray columns). To ensure that the uptake of the collagen fragments was indeed part of a degradation pathway, we also studied the effect of blocking lysosomal cysteine proteases in the fibroblasts. To this end, a similar experiment was set up with the inclusion of E-64d (Fig. 2B), an inhibitor that has been shown previously to block lysosomal degradation of internalized collagen-IV (28Kjoller L. Engelholm L.H. Hoyer-Hansen M. Dano K. Bugge T.H. Behrendt N. Exp. Cell Res. 2004; 293: 106-116Crossref PubMed Scopus (88) Google Scholar). Comparison of parallel samples obtained in the absence and presence of E-64d revealed that the blocking of intracellular degradation led to a marked increase in intracellular radioactivity in the uPARAP+/+ cells (Fig. 2B, black columns). Whereas this was the case both for intact collagen (columns 1 and 3) and cleaved collagen (columns 5 and 7), the amount of collagen fragments accumulated in the cells was much larger than the amount of intact collagen, also in the presence of the inhibitor. The presence of E-64d did not affect collagen uptake in uPARAP-/- cells (gray columns). To enable the visualization of the collagen fragments after endocytosis, FITC-labeled collagen type-I was treated with collagenase-3, leading to complete cleavage into fluorescent products with an electrophoretic migration pattern indistinguishable from that obtained with unlabeled collagen (Fig. 1A, lanes 3 and 4). The fluorescent fragments, as well as FITC-labeled intact collagen, were added to live fibroblasts from wild-type mice or littermate uPARAP-deficient mice, after which endocytosis was allowed to proceed in the presence of E-64d (Fig. 3). Examining the wild-type cells by fluorescence microscopy after 22 h of endocytosis, a strong fluorescence was observed in intracellular vesicles in the cells incubated with the cleaved FITC-collagen (Fig. 3B). The same cellular distribution was observed with FITC-labeled intact collagen-I (Fig. 3D), but strikingly, the amount of accumulated, fluorescence-labeled intact collagen was much lower than that of the collagen fragments, although the amounts of added fluorescent protein were identical. Again, this endocytosis was completely uPARAP-dependent, because no uptake or intracellular accumulation occurred with uPARAP-deficient cells (Fig. 3, A and C). The perinuclear, vesicular distribution of fluorescent protein, obtained with both cleaved and intact collagen-I, was indistinguishable from that obtained with fluorescence-labeled collagen-IV (result not shown), confirming results reported previously where the latter ligand was shown to accumulate in lysosomes in response to the arrest of lysosomal degradation (28Kjoller L. Engelholm L.H. Hoyer-Hansen M. Dano K. Bugge T.H. Behrendt N. Exp. Cell Res. 2004; 293: 106-116Crossref PubMed Scopus (88) Google Scholar). Altogether, these results showed that the defined proteolytic fragments of collagen-I are taken up much more efficiently than intact collagen by a uPARAP-dependent mechanism, through which they are directed to further lysosomal degradation. uPARAP-mediated Binding of Native and Cleaved Collagen in a Purified System—Molecular binding studies with collagen fragments and recombinant uPARAP were performed by s