H.

Engelholm
Thomas H. Bugge, Niels Behrendt and Lars
A Madsen, Peter Garred, Sven Burgdorf,
Amanda Moyer, Christian Honore, Charlotte
Jurgensen, Maria C. Melander, Lars Kjoller,
Daniel H. Madsen, Signe Ingvarsen, Henrik J.

uptake: a distinct degradation pathway
The non-phagocytic route for collagen
Regular Paper:
published online June 7, 2011 J. Biol. Chem.

10.1074/jbc.M110.208033 Access the most updated version of this article at doi:

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1

THE NON-PHAGOCYTIC ROUTE OF COLLAGEN UPTAKE: A DISTINCT DEGRADATION
PATHWAY

Daniel H. Madsen
1
, Signe Ingvarsen
1,2
, Henrik J. Jrgensen
1
, Maria C. Melander
1
, Lars Kjller
1,6
,
Amanda Moyer
3
, Christian Honor
4
, Charlotte A. Madsen
1
, Peter Garred
4
, Sven Burgdorf
5
, Thomas
H. Bugge
3
, Niels Behrendt
1
, Lars H. Engelholm
1


From the Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
1
, the Department of Biology, University
of Copenhagen, Copenhagen, Denmark
2
, Oral and Pharyngeal Cancer Branch, National Institute of Dental
and Craniofacial Research, National Institutes of Health, Bethesda, MD
3
, Laboratory of Molecular Medicine,
Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark
4
and Life and Medical Science
(LIMES) Institute, University of Bonn, Germany
5
.
6
Present address: LEO Pharma, Ballerup, Denmark

Corresponding author: Lars H. Engelholm, The Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter,
Ole Maaloes Vej 5, DK-2200, Copenhagen, Denmark.
Tel: +45 3545 6045. Fax: +45 3545 3797. E-mail: [email protected]

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 the cellular
internalization and degradation of large, soluble
collagen fragments, generated by initial cleavage
of 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 beta1-integrins, being responsible for
collagen phagocytosis, and the mannose receptor.
We have now utilized a newly developed
monoclonal antibody against uPARAP/Endo180,
which downregulates 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.
Beta1-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.

Remodeling of the extracellular matrix (ECM) is
required for a wide range of physiological and
pathological conditions such as morphogenesis,
organ growth, wound healing, arthritis, fibrosis and
tumor growth and metastasis (1-4). Collagens are
the most abundant components of the ECM with
collagen type I as the quantitatively dominating
subtype. Thus, collagen constitutes about 25-30% of
the dry weight of a human (5). The collagen in the
body is undergoing continuous renewal and
normally the collagen turnover rate is carefully
controlled. Depending on the tissue type or
extraneous events the collagen turnover rate can
change dramatically. Therefore, highly efficient
biological systems are needed in both the formation
and degradation of collagen throughout life.
In normal healthy tissue, collagen is fully
hydroxylated and forms insoluble, cross-linked
fibers and sheets of triple helical structures that are
resistant to attack by most proteases (6). A number
of proteases are nevertheless potentially capable of
initiating the collagen degradation process through
the cleavage of intact collagen fibers. These
proteases are the matrix metalloproteinases (MMPs)
MMP-1, MMP-2, MMP-8, MMP-13, MMP-14,
MMP-15 and MMP-16 and the cysteine protease
cathepsin K (7-9). So far, most studies of collagen
turnover have focused on the extracellular collagen
degradation mediated solely by these proteases.
Recently, however, we have established an
important interplay between the extracellular
collagenases and a collagen degradation pathway
http://www.jbc.org/cgi/doi/10.1074/jbc.M110.208033 The latest version is at
JBC Papers in Press. Published on June 7, 2011 as Manuscript M110.208033
Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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that involves endocytic uptake and intracellular
degradation of well-defined collagen fragments. In
particular, we have demonstrated that the collagen
fragments generated through the initial MMP-
mediated cleavage of collagen fibers can be
efficiently endocytosed and degraded in the
lysosomes in a process mediated by the specific
endocytic receptor, the urokinase plasminogen
activator receptor associated protein
(uPARAP)/Endo180. This sequential degradation
mechanism is required for complete and efficient
collagen turnover by mouse skin fibroblasts (10,11).
We have also demonstrated that the uptake of
solubilized, intact collagen mimics the efficient
uptake of the defined MMP-generated collagen
fragments (11). In addition to this composite
collagen degradation pathway involving non-
phagocytic collagen internalization, an alternative
collagen uptake mechanism also exists. This latter
pathway is dependent on beta1-integrin activity and
concerns the phagocytic uptake of large collagen
particles (12-14). At present, the elucidation of the
exact role of the different collagen receptors is an
important issue.

Through the generation of uPARAP/Endo180
knockout mice, we have previously demonstrated
that uPARAP/Endo180 is an indispensable
component for the internalization and lysosomal
degradation of solubilized collagen by primary
mouse fibroblasts, chondrocytes, and osteoblasts
(10,15,16). The uptake mechanism of
uPARAP/Endo180 has been shown to function
through constitutively recycling, clathrin-mediated
endocytosis (17,18).
Even though phenotypic studies of unchallenged
uPARAP/Endo180 knockout mice have only
revealed rather modest phenotypic consequences
((16) and Madsen et al., unpublished), lack of
uPARAP/Endo180 can lead to a dramatic collagen
accumulation in pathological conditions with high
collagen turnover such as cancer (19), thereby
confirming a role of uPARAP/Endo180 in the
process of ECM turnover in vivo. This role of
uPARAP/Endo180 was further substantiated by the
recent identification of a uPARAP/Endo180 null
mutation in cattle, which leads to a severe syndrome
characterized by dramatic bone defects (20). These
studies demonstrate the importance of the
uPARAP/Endo180-dependent collagen degradation
pathway in vivo and illuminate the need for further
investigations of this mechanism.

uPARAP/Endo180 is expressed in a variety of
different tissues (21,22) but a high
uPARAP/Endo180 expression is in particular
observed at sites of tissue remodeling, such as in the
osteoblasts and chondrocytes of the developing
bones (16,23,24). uPARAP/Endo180 is frequently
upregulated in invasive cancers (25-29), and most
often expression is observed in the stromal cells.
However, the total spectrum of cell types expressing
uPARAP/Endo180 is not yet known and it is also
unclear whether in all cases the receptor is active in
and required for collagen turnover (30).

In this paper, we describe an investigation of the
uptake of solubilized collagen in several cell types
of human and murine origin. By preventing collagen
uptake with a highly efficient mAb against
uPARAP/Endo180 and using cells with deficiency
for this and other collagen receptors, we can now
define the non-phagocytic pathway of collagen
degradation as a distinct mechanism in molecular
terms. Depending on the cell type, this pathway is
governed by either uPARAP/Endo180 or by another
member of the same protein family, the mannose
receptor.

Experimental Procedures

Reagents- The monoclonal mouse anti-
uPARAP/Endo180 antibodies (mAbs) 5f4 and
2h9F12 were raised against purified recombinant
soluble uPARAP/Endo180 and produced as
previously described (28). Isotype-matched control
anti-TNP antibody was produced as described in
(31). The following proteins were purchased from
the commercial sources indicated: acid-extracted
collagen type I from rat tail collagen, monoclonal
antibody against murine beta1-integrin (clone
HA2/5) and PE-conjugated anti-CD206 antibody
(BD Biosciences, Franklin Lanes, NJ ), Oregon
Green labeled gelatin, Oregon Green labeled
collagen type IV, holo-transferrin and cysteine
protease inhibitor E64d (Calbiochem, San Diego,
CA), interleukin 1 and tumor necrosis factor-
(Peprotech, Rocky Hill, NJ ), iodine-125 (Perkin
Elmer, Waltham, MA), monoclonal antibody
against human beta1-integrin (clone 4B4) (Beckman
Coulter, Brea, CA), cyclic RGD-peptide (Peptides
International, Louisville, KT), mannose-BSA
(Dextra Laboratories, Reading, United Kingdom),
CellMask Orange (Invitrogen, Carlsbad, CA, USA),
granulocyte macrophage colony-stimulating-factor
(GM-CSF) (Sigma-Aldrich, St. Louis, MO), FITC-

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conjugated anti-mouse antibody (DAKO, Glostrup,
Denmark) and proteinase K (Roche, Basel,
Switzerland).

Cultured cells- For the generation of human
macrophages, monocytes were isolated from human
blood as described (32). The monocytes were
seeded in the wells of a 24-well cell culture plate
and differentiated into macrophages using
granulocyte macrophage colony-stimulating-factor
(GM-CSF) at a concentration of 5 ng/ml over a 7-
day span. The cells were cultured in AIM-V
supplemented with 10% FCS and medium was
replenished on day 4.
Animal experiments were approved the NIDCR
Animal Care and Use Committee. For the isolation
of activated murine macrophages, wildtype and
littermate mannose receptor-/- mice in C57BL/6J
background (33) were injected intraperitoneally
with 2 ml of a 2 % Brewers TG solution (BD
Biosciences, Franklin Lanes, NJ ). Mice were killed
after 72 hours and peritoneal cells were collected by
lavage using 10 ml cold PBS. The lavage fluid was
centrifuged, the supernatant aspirated, and the cell
pellet resuspended in complete RPMI 1640 medium
with 10% DMSO, and the cells were frozen for later
use. Cells were then frozen directly for later use.
After thawing, cells were seeded in the wells of a
24-well cell culture plate in RPMI with 10% FCS
and 1% penicillin/streptomycin, and the adherent
cells were used for analysis.
The following established cell lines were purchased
from the indicated sources or from sources
described previously: Human gingival fibroblast
cell line HGF-1 (ATCC number CRL-2014), murine
sarcoma cell line NCTC 2472 (ATCC number CCL-
11), human osteosarcoma cell line MG63 (ATCC
number CRL-1427), murine fibroblast cell line
NIH/3T3 (ATCC number CRL-1658), human
prostate carcinoma cell line LNCaP (ATCC number
CRL-1740), human lung fibroblast cell line MRC-5
(ATCC number CCL-171), murine melanoma cell
line B16F10 (ATCC number CRL-6475) and human
colorectal adenocarcinoma cell line CaCo-2 (ATCC
number HTB-37) were all purchased from
American Type Culture Collection, ATCC,
Manassas, VA. Human fibrosarcoma cell line
HT1080, human rhabdomyosarcoma cell line RD
and the human cervix carcinoma cell line HeLa
(34), human breast adenocarcinoma cell line MCF-7
(35), human embryonic kidney cell line HEK-293
(36), murine Lewis Lung Carcinoma (LLC) (37),
human breast adenocarcinoma cell line MDA-MB-
231 (38). All cell lines were cultured according to
ATCC guidelines. Murine beta1-integrin deficient
fibroblasts (GD25) and GD25 fibroblasts re-
transfected with beta1-integrin (GD25-beta) were
kind gifts from Reinhard Fssler (39). Primary skin
fibroblasts were isolated from newborn
uPARAP/Endo180-/- or littermate wildtype
(uPARAP/Endo180+/+) mice as previously
described (15). Fibroblasts were cultured in DMEM
with 10% FCS and 1% penicillin/streptomycin.

Cell and organ extracts and Western blotting-
Cultured MG-63 cells and fibroblasts from
uPARAP/Endo180+/+ or littermate
uPARAP/Endo180-/- mice were lysed in a Triton
X-100 lysis buffer (10 mM Tris-HCl, 140 mM
NaCl, pH 7.4, 1 % Triton X-100, including
protease inhibitor cocktail III (Calbiochem)). Hearts
and lungs were isolated from uPARAP+/+mice or
littermate uPARAP-/- mice after perfusion of the
mice with 10 ml PBS. The organs were ground in
liquid nitrogen using a mortar and pestle and
homogenized in a CHAPS lysis buffer (0.1 M Tris-
HCl, 50 mM NaCl, pH 7.5, 2 % CHAPS, protease
inhibitor cocktail III) using a polytron homogenizer.
The cell lysates and organ homogenates were
clarified by centrifugation at 20,000 x g for 15 min.
at 4C. Protein concentrations were determined
using a BCA assay (Pierce) according to the
manufacturers instructions. SDS-PAGE was
performed with 15 g of cell lysate or organ
homogenate loaded in each well followed by
electroblotting onto PVDF membranes. For the
analysis of mAb 5f4 specificity, Western blotting
was performed with fibroblast lysates as previously
described (15) using mAb 5f4 at a final
concentration of 2 g/ml. For the analysis of organ
homogenates the PVDF membrane was incubated
with 20 ng/ml of
125
I-labeled mAb 5f4 at 4C
overnight followed by automated phosphorimager
analysis as previously described (11). Band
intensities were quantified using the Image Gauge
software (Fujifilm, Tokyo, J apan).

Endocytosis studies- Labeling of proteins with
125
I
and internalization studies of radiolabeled ligands
by cultured cells was performed essentially as
described in (15). All cells, except for macrophages,
were seeded in 24-well tissue culture plates at a
density of 1*10
5
cells/well. Macrophages were
seeded at a density of 2*10
5
cells/well. Cells were
cultured overnight to ensure maximal surface
adherence. For the functional blocking of

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uPARAP/Endo180 the monoclonal antibody (mAb)
5f4 was added to a concentration of 10 g/ml
(unless another concentration is indicated) during
this cell culture period. Isotype-matched control
antibody, anti-TNP, was likewise added during this
period. The cells were subsequently washed gently
in serum-free cell culture medium at 37C and were
then pre-incubated for 30 min. in assay buffer (the
appropriate cell culture medium with 20 mM Hepes,
pH 7.4 and 15 mg/ml BSA). In experiments with
functional blocking of uPARAP/Endo180, this
preincubation buffer included mAb 5f4 or negative
control antibody (anti-TNP) at a concentration of 10
g/ml (unless another concentration is indicated). In
experiments, which included functional blocking of
integrins, the preincubation buffer included 2 g/ml
or 10 g/ml of the indicated antibody or the
indicated concentration of cyclic RGD peptide. A
relatively short pre-incubation period in the
presence of integrin-blocking reagents (30 min,
without preceding overnight culture) proved
necessary in order to prevent detachment of the cells
(results not shown). In separate experiments, it was
ascertained that a similar short preincubation
procedure with 5f4 antibody (omitting the overnight
pre-culture with antibody) did not influence the
blocking efficiency markedly compared to
experiments with overnight preincubation (data not
shown). The assay buffer was removed and replaced
by assay buffer containing 133 ng/ml
125
I-labeled
protein and the indicated blocking reagents,
followed by incubation at 37C. The cells were
allowed to internalize ligand for 4-5 hours and then
washed three times with ice-cold PBS, and
incubated for <5 min in Trypsin-EDTA with 50
g/ml proteinase K to remove surface-bound
material. The detached cells were centrifuged at
1,000 g for 1 min at 4C, and the radioactivity in the
pellet (internalized material) and supernatant
(surface-released material) was measured in a
gamma counter. In each analysis, a maximum of 4
cell lines were examined and to minimize assay to
assay variations, MG63 cells were always included
as a reference cell line and measured values were
normalized to those of MG63. For each cell line the
collagen internalization was measured multiple
times. For the determination of the cellular protein
level of uPARAP/Endo180, the cells were incubated
with radiolabeled anti-uPARAP/Endo180 mAb
2h9F12 for 4 hours and the total amount of cell-
associated mAb 2h9F12 was measured.
Incubation of cultured cells with Oregon Green
labeled gelatin or collagen type IV followed by
fluorescence microscopy with a Zeiss LSM 510
microscope was performed as described previously
for fluorescence-labeled collagen type IV (10),
using an incubation period of 18-20 h in the absence
of protease-inhibitor or in the presence of 20 M
E64d. The cell surface of the murine fibroblasts was
stained as described in (10) using a monoclonal
antibody against uPAR and the cell surface of
MG63 cells was stained with 1 g/ml CellMask
Orange for 5 min before fixation of the cells.

Effect of antibody on uPARAP/Endo180 protein
level- Wildtype mouse primary skin fibroblasts or
human MG63 cells were seeded in 6-well plates at a
density of 6*10
5
cells per well. Cells were cultured
overnight at 37C, after which mAb 5f4 (10 g/ml)
was added. Control samples were cultured in the
presence of 10 g/ml isotype-matched control
antibody (anti-TNP). After 24 h, cells were washed
briefly in the wells with PBS with 5 mM EDTA,
followed by addition of a fresh aliquot (2 ml) of the
same buffer. After incubation for 30 min. at 37C,
cells were detached by pipetting in the same aliquot
of incubation buffer and transferred to Eppendorf-
tubes. The tubes were then centrifuged for 5 min at
400 x g. 80 l lysis buffer (10 mM Tris-HCl, 140
mM NaCl, pH 7.4 with 1 % Triton X-100 and
protease-inhibitor cocktail III) was added to each
cell pellet, followed by resuspension of cells. Tubes
were then placed on ice for 20 min, vortexed and
centrifuged for 20 min at 4C, 13,000 x g, after
which the supernatant was transferred to new tubes.
Protein concentrations were determined using a
BCA assay and equal protein amounts from each
sample of the supernatants were analyzed by
Western blotting using
125
I-labeled anti-
uPARAP/Endo180 antibody as described above,
except that mAb 2h9F12 was used. SDS-PAGE
analysis and Coomassie-staining was included to
ensure that equal protein amounts were indeed
loaded.

Collagen degradation assay- A collagen matrix was
reconstituted by polymerization and drying of acid-
extracted collagen type I as described in (40) with
inclusion of trace amounts of radiolabeled collagen
type I as described in (11). Fibroblasts were cultured
on the collagen-matrix in DMEM including 1 nM
interleukin 1 and 10 nM tumor necrosis factor- in
the absence of mAb 5f4 or in the presence of 50
g/ml mAb 5f4. After 5 days, the conditioned media

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was collected and analyzed by SDS-PAGE followed
by automated phosphorimager analysis. For the
generation of collagen fragments collagen type I
was cleaved with recombinant MMP-13 as
previously described (11).

Flow cytometry analysis of uPARAP/Endo180 and
mannose receptor expression- Dissociated cells
were washed twice in PBS-buffer with 5% FCS, and
incubated for 30 min, 4C in 5 ug/ml unlabeled
primary antibody or in 0.625 g/ml PE-conjugated
CD206 antibody. The cells were washed in PBS-
buffer containing 5% FCS and in the case of CD206
analyzed directly on a FACScalibur flow cytometer
(BD Biosciences, Franklin Lanes, NJ ). For the
analysis of uPARAP/Endo180 expression, cells
were washed as above and incubated for 30, 4C in
a 1:100 dilution of secondary FITC-conjugated anti-
mouse antibody. Cells were subsequently washed in
PBS-buffer + 5% FCS and analyzed by flow
cytometry.

RESULTS

Blocking collagen uptake through
uPARAP/Endo180. To enable the specific inhibition
of the uPARAP/Endo180-dependent collagen
uptake mechanism, a panel of monoclonal
antibodies (mAbs) was generated by immunization
of uPARAP/Endo180-deficient (-/-) mice with
recombinant human uPARAP/Endo180. The
utilization of mice with a deletion of the gene of
interest allows for the generation of mAbs capable
of recognizing human uPARAP/Endo180 as well as
the interspecies conserved parts of the protein (41-
43). We successfully generated eight mAbs against
human uPARAP/Endo180, of which seven cross-
reacted with the murine protein (results not shown).
The specificity of one of these antibodies,
designated 5f4, is shown in Fig. 1A. Both in
cultured fibroblasts (left panel) and mouse organ
extracts such as heart and lung (right panel), the
antibody displayed a very high specificity since
only one protein band was detected, migrating with
an apparent M
r
of 180 kDa. This protein was indeed
uPARAP/Endo180, since no protein was detected in
parallel samples derived from uPARAP/Endo180-/-
mice. The seven mAbs were tested for the ability to
inhibit the uptake of radiolabeled collagen type I by
primary mouse skin fibroblasts, a process reflecting
a central step in the combined proteolytic and
endocytic collagen degradation pathway (11,15).
One of them, mAb 5f4, proved able to very
efficiently reduce the collagen internalization
process (Fig. 1B). Although the assay conditions
with protease treatment of the cells did inevitably
leave a small amount of bound collagen on the
outside of the cells (see Methods), thus preventing a
complete isolation of the internalized radioactivity,
the antibody reduced the amount of radioactivity to
less than 25 %. Even more importantly, this level
was very close to that obtained with fibroblasts from
littermate uPARAP/Endo180 knockout mice. The
same antibody did not affect the cellular
internalization of holo-transferrin, an irrelevant,
non-collagen protein that is internalized through
clathrin-coated pits by means of the Transferrin
receptor (44) (Fig. 1C). Thus, mAb 5f4 does not
affect the general endocytosis machinery of the
cells. One additional antibody was also inhibitory
whereas the remainders were negative (results not
shown). The inhibitory property of mAb 5f4 was
further characterized and collagen internalization
experiments with mouse fibroblasts revealed an
approximate IC
50
of 0.04 g/ml (Fig. 1D), thus
indicating a very potent blocking reagent.
To directly visualize the effect of mAb 5f4 on the
resulting degradation process, a different
experimental system was applied, using
fluorescence-labeled collagens. Like collagen type I,
collagen type IV and gelatin (denatured collagen
type I) are internalized by fibroblasts in a
completely uPARAP/Endo180-dependent manner
and can be used in fluorescence-labeled form for
studies on endocytosis (10,11). uPARAP/Endo180
deficient (-/-) fibroblasts or littermate wildtype
(uPARAP/Endo180+/+) fibroblasts were incubated
with Oregon Green-labeled collagen type IV in the
absence or presence of mAb 5f4 and the cellular
uptake was monitored using fluorescence
microscopy (Fig. 2). In the absence of mAb 5f4,
internalized collagen was observed in
uPARAP/Endo180+/+ fibroblasts in vesicular
structures representing endosomes and lysosomes
(Fig. 2A). As expected, no or little internalized
collagen was found in the uPARAP/Endo180-/-
fibroblasts (results not shown). The intracellular
routing was indeed part of a degradation pathway
because an even more pronounced vesicular
accumulation of collagen was observed when the
lysosomal cysteine protease inhibitor E64d was
added (Fig. 2C). Both in the absence and presence
of E64d, this accumulation of collagen was
dramatically reduced when uPARAP/Endo180+/+
cells were incubated in the presence of mAb 5f4
(Fig. 2B and D), directly demonstrating the effect of

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this mAb on the total degradation pathway. An
antibody titration experiment with radiolabeled
collagen type IV (Fig. 2G) revealed that the uptake
of this type of collagen had the same high
sensitivity to mAb 5f4 as that noted above with
collagen type I.
To test whether mAb 5f4 also blocked collagen
turnover in human cells, we utilized the human
osteosarcoma cell line MG63, a cell line known to
express high levels of uPARAP/Endo180 (18,45).
An internalization experiment using radiolabeled
collagen type I revealed a blocking effect of mAb
5f4 with this human cell line that was similar to that
found with uPARAP/Endo180+/+mouse fibroblasts
and a similar IC
50
value was determined (Fig. 1E).
Furthermore, when fluorescence-labeled gelatin was
added to MG63 cells, the internalized protein
accumulated in the endosomal/lysosomal
compartments (Fig. 2E), whereas in the presence of
mAb 5f4, this accumulation was clearly inhibited
(Fig. 2F). Both with human and murine cells, even
very low concentrations of antibody were sufficient
to block the uptake of the fluorescence-labeled
ligand (Supplemental Fig. 1), in complete
accordance with the titration studies performed with
radiolabeled collagens. Thus, the uptake of all of the
collagen ligands tested in these cells displayed a
strong sensitivity to mAb 5f4 against
uPARAP/Endo180.

These observations raised the question about the
mechanism through which mAb 5f4 could
counteract the function of uPARAP/Endo180 in
collagen internalization. First, we utilized a purified
system with surface plasmon resonance to test any
blocking effect of the antibody against the direct
interaction between uPARAP/Endo180 and
collagen. Using an established BIAcore setup with
recombinant uPARAP/Endo180 immobilized on a
distinct catching monoclonal antibody, mAb
2h9F12 (11), we first ensured that this catching
antibody did not prevent the subsequent binding of
mAb 5f4 to uPARAP/Endo180 (results not shown).
In the next step, we injected solubilized collagen
type I into flow cells with immobilized uPARAP
that was either unoccupied or saturated with mAb
5F4 (Supplemental Fig. 2). Somewhat surprisingly,
this experiment showed that mAb 5f4 had no
inhibitory effect on collagen binding.

Consequently, we tested whether the antibody had
an influence on the cellular uPARAP/Endo180
protein level. Mouse primary fibroblasts and human
MG63 cells were cultured in the presence or
absence of mAb 5f4, after which the cellular level
of uPARAP/Endo180 was assessed by Western
blotting (Fig. 3). Strikingly, with both cell types,
cultivation in the presence of the antibody led to a
strongly reduced level of the collagen receptor.
Thus, the effect of mAb 5f4 is similar to that
observed with certain other antibodies against cell
surface receptors which function through an
apparent downregulation of their target antigen
(46,47), an effect likely to reflect an antibody-
induced failure of receptor recycling.

We have previously demonstrated that the
intracellular collagen turnover pathway is operative
when fibroblasts act to remodel an insoluble
collagen matrix. Thus, uPARAP/Endo180 is
essential for the clearance of the initial collagen
cleavage products that are released through the
action of MT1-MMP when fibroblasts invade a
trypsin-resistant collagen matrix (Fig. 4A) (11).
This clearance defect results in the accumulation of
defined collagen fragments in the conditioned media
of the uPARAP/Endo180-/- fibroblasts (Fig 4B,
lane 4) but not of the uPARAP/Endo180+/+
fibroblasts (Fig 4B, lane 3). Strikingly, incubation
of uPARAP/Endo180+/+fibroblasts with the mAb
5f4 led to the same accumulation of collagen
fragments that were observed for the
uPARAP/Endo180-/- fibroblasts (Fig 4B, lane 5).
This demonstrates that, also in a composite cell
culture assay of collagen degradation, it is possible
to mimic uPARAP/Endo180 deficiency by
incubation of uPARAP/Endo180+/+fibroblasts with
mAb 5f4.

The role of uPARAP/Endo180 in collagen
internalization by different cell types. A panel of
human and murine cell lines of epithelial or
mesenchymal origin (Table 1) were screened for
their ability to internalize radiolabeled collagen.
Simultaneously, for each cell line, the level of
uPARAP/Endo180 expression was determined
using a distinct, high affinity anti-
uPARAP/Endo180 mAb, designated 2h9F12. As
shown by previous BIAcore studies ((11) and
results not shown), the interaction between
uPARAP/Endo180 and this antibody is stable even
at low pH, thereby precluding endosomal
dissociation of the complex. Consequently, the total
amount of cell bound mAb 2h9F12 on different cell
types enables a determination of the relative
amounts of uPARAP/Endo180 molecules. For each

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cell line the collagen internalization was measured
and the collagen internalization could then be
plotted against the determined uPARAP/Endo180
levels (Fig. 5A).
Strikingly, this analysis revealed a strong
correlation between the uPARAP/Endo180
expression of the cells and their ability to internalize
collagen (p=0.00018). As expected, the
mesenchymal cell types proved much more efficient
in internalizing collagen than the epithelial cells
(Fig. 5A, empty dots). With the exception of
rhabdomyosarcoma (RD) cells, all the mesenchymal
cells were capable of efficient collagen
internalization (with a read-out of more than 40% of
the reference cell line MG63). The epithelial cell
lines expressed low levels of uPARAP/Endo180
(Fig. 5A, filled dots) and all of them were incapable
of efficient internalization of collagen. To make
sure that the uPARAP/Endo180 level correlated
with collagen internalization specifically, and not
just with the general endocytic activity of the cells,
we also examined the uptake of holo-transferrin
(Fig. 5B). This analysis did not reveal any
correlation between uPARAP/Endo180 level and
the general endocytic activity of the cell lines.

To examine whether uPARAP/Endo180 also on
these cell types was directly responsible for the
collagen uptake, the blocking uPARAP/Endo180
mAb 5f4 was utilized. Internalization of
radiolabeled collagen type I was carried out in the
presence or absence of mAb 5f4. In the presence of
mAb 5f4, the collagen uptake was markedly
reduced for all the cell lines capable of efficient
collagen internalization (Fig. 5). As mentioned
above, the mesenchymal RD cells expressed low
levels of uPARAP/Endo180 and internalized low
levels of collagen. The presence of mAb 5f4 did not
reduce collagen internalization for this cell line.
Of the epithelial cell lines, HeLa cells had the
highest level of uPARAP/Endo180 expression, and,
in fact, this epithelial cell line was the only one that
displayed reduced collagen internalization in the
presence of mAb 5f4.

Beta1-integrins are not involved in the endocytosis
of soluble collagen. Although collagen
internalization was found to be a completely
uPARAP/Endo180-dependent process in our
experimental systems, we could not exclude the
possibility that other receptors could be involved as
well. Beta1-integrins are key mediators of cellular
adhesion to collagen (48) and are required for the
phagocytic uptake of collagen-coated beads (49).
Therefore, and because a central issue to be
elucidated is the distinction between the present
route of collagen degradation and the phagocytic
uptake of particulate collagen, we studied the
possible function of beta1-integrins in the
internalization of solubilized, radiolabeled collagen.
This was examined using beta1-integrin null mouse
fibroblasts (GD25), as well as GD25 fibroblasts re-
transfected with beta1-integrin (GD25-beta) (fig
7A). Both cell lines were capable of internalizing
solubilized collagen and furthermore, collagen
internalization could be markedly reduced by the
presence of mAb 5f4 for both cell lines. Thus, also
with these cells uPARAP/Endo180 has a decisive
function in the collagen uptake mechanism. Beta1-
integrin null fibroblasts (GD25) internalized
collagen very efficiently (155% of MG63 cells
under our experimental conditions). The efficient
internalization in GD25 cells clearly demonstrated
that beta1-integrins are not required for the uptake
of solubilized collagen. Indeed, with the present
fibroblast strains, the beta1-integrin re-transfected
(GD25-beta) cells even internalized less collagen
than GD25 cells. This difference in collagen uptake
between the GD25 and the GD25-beta fibroblasts
could be explained by the different levels of
uPARAP/Endo180 expression (Fig. 7B) and was
not caused by a general difference in their endocytic
capacities (Fig. 7C).
To examine the possibility that beta1-integrin
independent collagen endocytosis was restricted to
murine fibroblasts we analyzed the human
osteosarcoma cell line MG63 (Fig. 7D). To
investigate the role of beta1-integrin for collagen
internalization in a human cell line we utilized the
blocking beta1-integrin antibody 4B4 (12,13,50).
Collagen internalization by MG63 cells was not
affected by the addition of 4B4 (Fig. 7D, columns 3
and 4). To confirm this result we also utilized cyclic
RGD peptide (13,36,51) to block beta1-integrin. In
the presence of cyclic RGD peptide collagen
internalization was likewise unaffected (Fig 7D,
columns 5 and 6), demonstrating that also the
human cell line MG63 is capable of beta1-integrin
independent collagen endocytosis.
Beta1-integrin dependence was also tested for the
murine fibroblast cell line NIH/3T3 using blocking
antibody and cyclic RGD peptide. This cell line was
included in the analysis because of reported beta1-
integrin mediated phagocytic collagen uptake by
this cell line (52,53). However, in our assay of non-
phagocytic collagen internalization we did not

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observe any beta1-integrin dependence for this cell
line (results not shown).

The mannose receptor is responsible for collagen
endocytosis in macrophages. In addition to
fibroblasts, osteoblasts and chondrocytes, which are
key players in collagen turnover in the healthy body
(16,54,55), macrophages play a decisive role in the
matrix degradation processes in inflammatory
conditions (3,56,57). Interestingly,
uPARAP/Endo180 expression has in certain cases
been demonstrated on macrophages (58-60). This
raised the question whether an uptake mechanism
for solubilized collagen exists in macrophages and
whether any such mechanism depends on
uPARAP/Endo180 or different receptors. To
examine the process of collagen endocytosis in
macrophages, we isolated monocytes from human
plasma and differentiated these cells to
macrophages in vitro by cell culture for 8 days in
the presence of GM-CSF. No uPARAP/Endo180
was detected on the undifferentiated monocytes,
whereas a low level of uPARAP/Endo180
expression could indeed be observed after
differentiation to macrophages (Supplemental Fig.
3). However, the amounts of uPARAP/Endo180
detected were very low as compared to MG63 cells
(Fig. 8A). The macrophages were assayed for their
ability to internalize collagen and in spite of the
very low level of uPARAP/Endo180 expression,
they were capable of internalizing collagen at levels
comparable with those of MG63 cells (Fig. 8B).
Addition of mAb 5f4 did not affect collagen
internalization significantly (Fig. 8B, column 4),
indicating that the macrophages were taking up the
collagen in a process independent of
uPARAP/Endo180. Interestingly, this process was
also insensitive to blocking of beta1-integrins, as
shown with the same reagents as used above (Fig.
8C). To confirm that the internalized collagen was
routed to the lysosomes for degradation, we
repeated the collagen internalization assay with
inclusion of E64d (Fig. 8D). We have previously
shown that this inhibitor retards the lysosomal
degradation of collagen which, in the case of murine
fibroblasts, leads to an approximately 2-fold
increase in the amount of intracellular collagen
under our experimental conditions (11). In the case
of macrophages, addition of E64d led to an
approximately four-fold increase in the amount of
intracellular collagen. This large effect could be
indicative of a high lysosomal collagen degradation
activity in macrophages in the absence of inhibitor.
Various recent studies have demonstrated that the
mannose receptor (MR), which belongs to the same
receptor family as uPARAP/Endo180, is able to
bind to collagen and to promote the uptake of
solubilized fluorescent-labeled collagen (61,62).
Interestingly, the above-mentioned monocyte
differentiation process led to the expression of the
MR on the resulting macrophages, as verified by
flow cytometry analysis (Supplemental Fig. 3). To
examine whether macrophages utilize the MR to
internalize collagen, we isolated activated
macrophages from the peritoneum of MR deficient
mice (MR-/-) or littermate wildtype (MR+/+) mice.
First, we showed that the routinely used MR ligand
model, mannose-BSA, was internalized exclusively
by the MR+/+macrophages and not by the MR-/-
macrophages (Fig. 8F), demonstrating that MR-
dependent endocytosis processes are indeed ongoing
during our standard assay conditions. Strikingly, in
the subsequent collagen internalization assays, the
MR-/- macrophages displayed a markedly reduced
ability to internalize collagen relative to the MR+/+
cells (Fig. 8E), indicating a dominant function of
MR in collagen endocytosis in this cell type. In
contrast, neither MR+/+ nor MR-/- macrophages
were affected by addition of mAb 5f4 (Fig. 8E).

Altogether, our results show that members of the
receptor family comprising uPARAP/Endo180 and
MR are dominant mediators of the endocytic uptake
of solubilized collagen, with the identity of the
active receptor differing between different cell
types.

DISCUSSION

In this study we have examined the endocytosis of
solubilized collagen in a panel of mesenchymal and
epithelial cell lines and in human and murine
macrophages. We have previously demonstrated
that this endocytic process models the non-
phagocytic uptake of the primary collagen cleavage
products ( and fragments), that are
generated by the action of collagenolytic proteases
in an organized degradation pathway (11). To
enable analysis of the specific role of the collagen
internalization receptor uPARAP/Endo180 in all of
the cell types studied, we developed a monoclonal
antibody against uPARAP/Endo180 that works to
deplete the cells for the receptor. This antibody,
designated mAb 5f4, very efficiently prevents the
uPARAP/Endo180-dependent collagen uptake in
both murine and human cells.

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The analysis of a range of established cell lines
revealed a striking correlation between the cellular
collagen internalization capability and the cellular
protein level of uPARAP/Endo180. For all cell
lines, which displayed efficient collagen
internalization, the addition of mAb 5f4 led to a
pronounced reduction in the amount of internalized
collagen. The epithelial cell lines internalized much
less collagen than the mesenchymal cells and they
expressed lower amounts of uPARAP/Endo180.
Among the epithelial cell lines, HeLa cells
displayed the highest expression level of
uPARAP/Endo180 and interestingly, this cell line
did internalize less collagen in the presence of mAb
5f4 and was the only epithelial cell type with this
property.

Among the examined cell types we found that the
gingival fibroblast cell line HGF-1 expressed the
highest protein level of uPARAP/Endo180. As
expected, this cell line displayed efficient and
uPARAP/Endo180-dependent collagen
internalization. An altered collagen metabolism by
gingival fibroblasts is known to be involved in such
pathological conditions as periodontal disease and
gingival overgrowth, which are characterized by
increased and decreased collagen degradation,
respectively. In this cell type, it has long been
recognized that intracellular collagen degradation is
a central route of turnover, but so far, only integrins
have been suggested as mediators of this
degradation pathway (12,49,63). Our study reveals
that uPARAP/Endo180 is required for the
endocytosis of collagen by gingival fibroblasts, thus
potentially opening new therapeutic options for
future treatment of periodontal disease.

The distinction between different mechanisms of
collagen uptake and the identification of the
receptors involved are two important issues. The
beta1-integrins are an additional type of collagen
receptors, which have been shown to be involved in
processes of collagen internalization (12,49,53).
However, those studies have focused entirely on a
phagocytic uptake mechanism, i.e. the uptake of
collagen-coated beads. A wide range of different
cell types express beta1-integrins (12,48,53),
including many of the cell lines that we have found
in this study to be capable of non-phagocytic,
uPARAP/Endo180-dependent collagen uptake.
Thus, some cell lines appear to possess the ability to
internalize collagen in both a phagocytic and a non-
phagocytic manner, notably including gingival
fibroblasts and NIH-3T3 cells.

However, although our blocking studies with mAb
5f4 on the non-phagocytic pathway did document a
central role of uPARAP/Endo180, they did not
exclude that beta1-integrins could also be involved
in this collagen uptake process, since the two
receptors might cooperate. To study this possibility,
we examined the non-phagocytic collagen uptake by
fibroblasts derived from beta1-integrin deficient
mice (39). We found that these fibroblasts, termed
GD25, displayed efficient collagen internalization
and that this uptake was completely
uPARAP/Endo180-dependent. That beta1-integrins
were indeed not involved in the uptake of collagen
in our assay system was further substantiated by the
fact that GD25 fibroblasts re-transfected with beta1-
integrin (GD25-beta) (64) actually internalized less
collagen than the GD25 fibroblasts. This difference
is most likely a consequence of clonal variations
between the two cell lines, resulting in different
levels of uPARAP/Endo180 expression. The
analyses using GD25 fibroblasts are in line with
previous observations where antibody-mediated
blocking of beta1-integrin on murine skin
fibroblasts did not affect the uptake of solubilized
collagen (15).

The distinction between these mechanisms of
collagen internalization is supported by a recent
study where Shi et al. examined the uptake of
soluble collagen and of insoluble polymerized
collagen (14). These authors found that beta1-
integrins are not involved in the endocytosis of
soluble collagen, in agreement with our results,
whereas they are indeed dominant mediators of the
internalization of the collagen originating from an
insoluble collagen-matrix, in consistence with the
studies cited above on collagen phagocytosis.
Furthermore, Shi et al. found that
uPARAP/Endo180 is indeed also involved in a
particular turnover mechanism for insoluble matrix-
collagen that is sensitive to matrix metalloprotease
inhibitors (14). This points to a sequential
degradation mechanism for matrix-collagen in
agreement with our previous studies (11). Our
present work suggests that this pathway may be a
widespread mechanism in many cell types.

The existence of two distinct mechanisms for
collagen uptake leads to the question about the role
of these two collagen degradation pathways in vivo.

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Intracellular degradation of collagen has been
known for several decades to be operative in vivo in
a number of physiological and pathological contexts
(63) but the identity of the responsible receptors has
been limited to speculation. Recently, however, a
number of studies have confirmed that
uPARAP/Endo180 does indeed take part in collagen
turnover processes in vivo. Crooked Tail Syndrome
is a severe genetic disease observed in the inbred
Belgian Blue strain of cattle, which results in
massive bone defects that necessitate the
euthanization of the affected animals. In a recent
genetic study, the underlying cause of the disease
was identified as a frame-shift mutation in the gene
encoding uPARAP/Endo180, leading to the
expression of a non-functional protein (20).
Expression of uPARAP/Endo180 has also been
demonstrated during bone development in normal
mice (23) but in uPARAP/Endo180 knockout mice
in the absence of challenge, only a slightly reduced
bone length has been observed ((16) and Madsen et
al., unpublished). However, in combination with
deficiency for MT1-MMP, an important
collagenolytic protease, uPARAP/Endo180
deficiency was found to lead to fatal bone defects
(16). Thus, the collagen turnover processes of
uPARAP/Endo180 play a central role in bone
development in mice as well as in cattle.
In a pathological context, uPARAP/Endo180
expression has been observed in the tumor stroma of
multiple types of cancer, including breast cancer
(27). The functional implication of
uPARAP/Endo180 in breast cancer progression was
investigated using a mouse model of genetically
induced mammary cancer (MMTV-PymT model)
(19). In this cancer model, uPARAP/Endo180
deficiency leads to delayed growth of the tumors
and, interestingly, also to large accumulations of
intratumoral collagen, demonstrating that
uPARAP/Endo180 participates in the turnover of
collagen in this pathological condition.

uPARAP/Endo180 belongs to a protein family
comprising three other receptors, the mannose
receptor (MR), phospholipase A2 receptor (PLA2R)
and DEC-205. All four receptors share a common
protein domain composition, which includes a well-
conserved fibronectin type II domain (65,66). This
domain is believed to be the most important element
for the interaction between uPARAP/Endo180 and
collagen. MR has for many years been linked to the
innate immune system as a receptor capable of
binding to carbohydrates on the surface of certain
pathogens, thereby promoting their phagocytosis
(67). Recently, however, MR has also been reported
to bind and internalize collagen in vitro (61,62) and
to be responsible for the clearance of denatured
collagen from the circulation in vivo (68). MR is
primarily expressed by macrophages but is also
observed on certain other cell types such as the
specialized endothelial cells of the liver sinusoids
(67,69).

Macrophages are known to be involved in
promoting the degradation of the extracellular
matrix in multiple pathological processes
(3,56,57,70). In this work we have examined
collagen endocytosis by macrophages to study the
relative role of uPARAP/Endo180 and MR in the
non-phagocytic collagen degradation pathway. The
differentiation of human blood monocytes to
macrophages in vitro led to a dramatic upregulation
of MR and also a moderate upregulation of
uPARAP/Endo180. Further determination of the
cellular level of uPARAP/Endo180 did, however,
reveal that the macrophages expressed less than
20% of the reference cell line MG63. Strikingly,
these macrophages proved capable of efficient
collagen internalization, but were insensitive to
addition of mAb 5f4 against uPARAP/Endo180.
Previous studies using fluorescence-labeled
solubilized collagen suggest that MR is the receptor
responsible for collagen internalization in the case
of macrophages (62). To clarify whether this was
also the case in our assay system, we isolated
peritoneal macrophages from MR-/- mice or from
littermate MR+/+ mice. Collagen internalization
assays with these primary cells revealed a clear
dependency on MR for collagen internalization. J ust
like the situation observed with mesenchymal cells,
we did not observe any beta1-integrin dependence
for the collagen uptake by cultured macrophages.
Altoghether, these observations suggest that the
mannose receptor could be critical for the
degradation of collagen in pathological conditions,
which involve increased macrophage influx and/or
activation. Such conditions include cancer, fibrosis
and arthritis (3,57,70).
Subsets of macrophages have been identified as
uPARAP/Endo180 positive in vivo in certain
locations (58-60). To this end, we cannot rule out
the possibility that our cultured macrophages
represent a certain subtype of macrophages, whereas
other subtypes of macrophages could display a
higher level of uPARAP/Endo180 expression and

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11

be capable of internalizing collagen in a
uPARAP/Endo180-dependent manner.

This study reveals that uPARAP/Endo180 and the
mannose receptor are two major mediators of the
non-phagocytic collagen degradation pathway, with
uPARAP/Endo180 acting primarily on
mesenchymal cells and MR on macrophages. It
remains a possibility that the other members of the
same protein family, PLA2R and DEC-205 could
have a similar function on other cell types. In fact,
the PLA2R has been found to interact with collagen
in vitro (71) and we speculate that this interaction
could likewise be connected to intracellular
degradation of collagen. Altogether, our work points
to a previously underestimated role of the mannose
receptor protein family members in the turnover of
extracellular matrices. Since these breakdown
processes are important in malignant as well as non-
malignant diseases this concept opens a new range
of therapeutic strategies.

Acknowledgements- The excellent technical
assistance of Suzanne K. Mller and Katharina H.
Stegmann is gratefully acknowledged.

This work was supported by the Danish Cancer
Society, the Danish Medical Research Council, the
Danish Cancer Research Foundation, the Lundbeck
Foundation, the Danish National Research
Foundation (Danish-Chinese Center for Proteases
and Cancer) and the European Communitys
Seventh Framework Programme FP7/2007-2011
under grant agreement n201279 (N. B.), by the
Lundbeck Foundation and the Grosserer Alfred
Nielsen og Hustrus foundation (L. H. E.) and by
the NIDCR Intramural Research Program (A. M.
and T. H. B.). The work was supported by personal
grants from Copenhagen University Hospital (D. H.
M. and H. J. J.) and from the University of
Copenhagen, Faculty of Science (S. I.).



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FOOTNOTES

The abbriviations used are: ECM, extracellular matrix; uPARAP, urokinase plasminogen activator receptor-
associated protein; MMP, matrix metalloprotease; MT1-MMP, membrane type 1 matrix metalloprotease;
mAb, monoclonal antibody; TNP, tri-nitrophenyl; PE, phycoerythrin; BSA, bovine serum albumin; FITC,
fluorescein isothiocyanate; MR, mannose receptor; MMTV-PymT, mouse mammary tumor virus polyoma
middle T; PLA2R, phospholipase A2 receptor.

FIGURE LEGENDS

Table 1. Established cell lines included in the study of collagen uptake.

Fig. 1. anti-uPARAP/Endo180 mAb 5f4 inhibits cellular collagen internalization. A, mAb 5f4 specifically
recognizes uPARAP/Endo180. Detergent extracts of cultured fibroblasts isolated from newborn mice (left
panel) or mouse heart and lung (right panel) were analyzed by Western blotting, using mAb 5f4 as the
primary antibody. In each panel, samples from wildtype (uPARAP+/+) and uPARAP/Endo180 deficient
(uPARAP-/-) mice were run in parallel lanes. The electrophoretic mobility of M
r
marker proteins is indicated
to the left. uPARAP/Endo180 migrates with an apparent M
r
of 180 kDa. B, mAb 5f4 inhibits collagen
internalization by murine fibroblasts. Primary fibroblasts from wildtype (uPARAP/Endo180+/+) mice (dark
grey columns) or littermate uPARAP/Endo180-/- mice (light grey columns) were allowed to internalize
radiolabeled soluble collagen I for 4 hours in the absence of antibody or in the presence of 10 g/ml mAb
5f4 (hatched columns). C, Anti-uPARAP/Endo180 mAb 5f4 does not affect internalization of holo-
transferrin. Murine fibroblast cultures were assayed as in B, but using radiolabeled holo-transferrin instead of
collagen I. D, Anti-uPARAP/Endo180 mAb 5f4 inhibits collagen internalization in a dose-dependent
manner. Murine primary fibroblast cultures were allowed to internalize radiolabeled soluble collagen I in the
absence of antibody, in the presence of mAb 5f4 at concentrations ranging from 0.01 g/ml to 100 g/ml or
in the presence of 100 g/ml of a monoclonal isotype matched irrelevant control antibody (anti-TNP).
uPARAP/Endo180-/- fibroblasts (light grey column) were included in the analysis for comparison. A half-
maximum inhibition was obtained with an approximate concentration of 0.04 g/ml of mAb 5f4. Both in B,
C and D, the internalized radioactivity in untreated uPARAP/Endo180+/+fibroblasts was defined as 100%
internalization. E, Anti-uPARAP/Endo180 mAb 5f4 inhibits collagen internalization by human MG63 cells.
An experiment was performed as in B, but using the human osteosarcoma cell line MG63 instead of primary
murine fibroblasts. Internalization by MG63 cells in the absence of antibody was defined as 100%
internalization. Note that a similar inhibitory efficacy of mAb 5f4 was observed with human and mouse
cells. For B-E, all samples were analyzed in triplicates. Error bars indicate standard deviations.

Fig. 2. Anti-uPARAP/Endo180 mAb 5f4 prevents the accumulation of collagen in intracellular vesicles. A-
D, The intracellular accumulation of fluorescent-labeled collagen type IV in murine fibroblasts is prevented
by anti-uPARAP/Endo180 mAb 5f4. Oregon Green labeled collagen IV was added to primary fibroblast
cultures derived from uPARAP/Endo180+/+mice, after which the cells were cultured for 18 hours in the
absence of the lysosomal protease inhibitor E64d (A and B) or in the presence of 20 M E64d (C and D).
The internalization of collagen was carried out in the absence of antibody (A and C) or in the presence of 10

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g/ml mAb 5f4 (B and D). E-F, Anti-uPARAP/Endo180 mAb 5f4 prevents the intracellular accumulation of
fluorescent-labeled gelatin in the human osteosarcoma cell line MG63. Oregon Green labeled gelatin was
added to cultured MG63 cells in the presence of E64d. Internalization was carried out for 20 hours in the
absence of antibody (E) or in the presence of 10 g/ml mAb 5f4 (F). G, Cellular uptake of collagen IV is
equally sensitive to mAb 5f4 as the uptake of collagen I. The mAb 5f4 dose dependence of collagen IV
uptake was measured using radiolabeled collagen ligand. The conditions were the same as used in Fig. 1D,
except that the ligand was
125
I-labeled collagen IV.

Fig. 3. Downregulation of the uPARAP/Endo180 protein level by mAb 5f4. Primary mouse fibroblasts (A)
or human MG63 osteosarcoma cells (C) were cultured in the presence of mAb 5f4 or anti-TNP control mAb,
after which detergent lysates of the cells were analyzed by Western blotting using
125
I-labeled anti-
uPARAP/Endo180 mAb 2h9F12 (upper panels) or SDS-PAGE and Coomassie-staining as loading control
(lower panels). The quantification of band intensities in fibroblast lysates (A) is depicted in diagram B, and
the band intensities in MG63 lysates (C) is depicted in diagram D.

Fig. 4. Anti-uPARAP/Endo180 mAb 5f4 treatment mimics uPARAP/Endo180 deficiency in a composite
assay of collagen degradation. A, Schematic illustration of the experimental setup. A collagen matrix was
reconstituted by polymerization of acid-extracted collagen type I in which trace amounts of radiolabeled
collagen type I was included. Fibroblasts were cultured on top of the collagen matrix for 5 days after which
the conditioned culture supernatants were harvested and analyzed by non-reducing SDS-PAGE, followed by
phosphorimaging analysis of the polyacrylamide-gel for the localization of specific collagen fragments
released from the collagen matrix. B, Anti-uPARAP/Endo180 mAb 5f4 leads to extracellular accumulation
of collagen degradation products. The culture supernatants from the following cells are shown: lane 3,
Untreated uPARAP/Endo180+/+ cells; lane 4, untreated uPARAP/Endo180-/- cells; lane 5,
uPARAP/Endo180+/+cells cultured in the presence of 50 g/ml mAb 5f4. The following purified reference
proteins are shown: lane 1,
125
I-labeled, intact collagen type I; lane 2,
125
I-labeled, cleaved collagen type I.
The positions of 14 and 34 collagen degradation fragments are indicated at right.

Fig. 5. uPARAP/Endo180 expression correlates with collagen internalization in a variety of cell types. A,
Correlation between uPARAP/Endo180 expression and collagen internalization. Fifteen epithelial (filled
dots) and mesenchymal cell lines (empty dots), in addition to uPARAP/Endo180+/+and uPARAP/Endo180-
/- fibroblasts, were assayed for the expression of uPARAP/Endo180 and the ability to internalize
radiolabeled collagen. The level of uPARAP/Endo180 expression was determined as the amount of cell
bound radio-labeled anti-uPARAP/Endo180 mAb 2h9F12. Collagen internalization was assayed as in Fig. 1.
MG63 cells were used as a reference cell line for the normalization of both collagen internalization and the
level of uPARAP/Endo180 expression. Each cell line is represented as one point in the scatterplot, with the
relative uPARAP/Endo180 level assigned to the x-axis and the collagen internalization activity assigned to
the y-axis. A correlation analysis revealed a Pearson correlation coefficient r=0.77 and p=0.00018. A
regression line is included in the graph. B, No correlation between uPARAP/Endo180 expression and holo-
transferrin internalization. The same cell lines as in A were also assayed for the ability to internalize
radiolabeled holo-transferrin. MG63 cells were used as a reference cell line for the normalization of holo-
transferrin internalization. A scatterplot similar to the one in A was generated but with holo-transferrin
internalization assigned to the y-axis. All samples were analyzed in triplicates. Error bars indicate standard
deviations.

Fig. 6. uPARAP/Endo180 mediates collagen internalization in a variety of cell types. Collagen
internalization by a variety of established cell lines is inhibited by mAb 5f4. Collagen internalization was
measured for each cell line used in Fig 4. Internalization was carried out in the absence of antibody (black
columns) or in the presence of 10 g/ml of mAb 5f4 (grey columns). The cell lines are ranked in the diagram
according to their level of uPARAP/Endo180 expression, with the highest expressing cells at the left. MG63
cells were used as a reference cell line for the normalization of both collagen internalization and the level of
uPARAP/Endo180 expression. All samples were analyzed in triplicates. Error bars indicate standard
deviations.

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Fig. 7. Beta1-integrins are not involved in the internalization of soluble collagen. A, Murine beta1-integrin
null fibroblasts are capable of internalizing collagen. Murine beta1-integrin-deficient fibroblasts
(GD25)(black columns) and beta1-integrin retransfected fibroblasts (GD25-beta)(grey columns) were
analyzed for their ability to internalize radiolabeled soluble collagen I in the absence of antibody or in the
presence of 10 g/ml mAb 5f4 (hatched columns). The collagen internalization assay was performed as
described in Fig. 1. B, uPARAP/Endo180 expression correlates with collagen internalization efficiency. The
relative levels of uPARAP/Endo180 expression were determined for GD25 and GD25-beta fibroblasts as
described in Fig. 4. C, GD25 and GD25-beta fibroblasts have the same overall endocytic capacity. An
internalization assay was performed with radiolabeled holo-transferrin as in Fig. 1B. D, MG63 cells
internalize collagen in a beta1-integrin independent process. MG63 cells were analyzed for their ability to
internalize labeled soluble collagen I in the absence of any exogenous reagents (column 1), or in the presence
of 10 g/ml of mAb 5f4 (column 2), 2 g/ml anti-beta1 mAb 4B4 (column 3), 10 g/ml anti-beta1 mAb 4B4
(column 4), 50 M cyclic RGD peptide (column 5) or 200 M cyclic RGD peptide (column 6). A-D, MG63
cells were in all cases included in parallel analyses and used as a reference for the normalization of the data.
All samples were analyzed in triplicates. Error bars indicate standard deviations.

Fig. 8. The mannose receptor (MR) governs collagen internalization in macrophages. A-D, Human blood
monocytes were differentiated to macrophages in vitro. A, Cultured macrophages express low amounts of
uPARAP/Endo180. The relative level of uPARAP/Endo180 expression by cultured macrophages was based
on the amount of cell bound radio-labeled anti-uPARAP/Endo180 mAb 2h9F12 as in Fig. 4. MG-63 cells
were included as a normalization reference. B, Macrophages internalize collagen in a uPARAP/Endo180-
independent process. The internalization of radiolabeled collagen type I was measured for MG63 cells
(columns 1 and 2) and macrophages (columns 3 and 4) in the absence (columns 1 and 3) or presence
(columns 2 and 4) of 10 g/ml of mAb 5f4. Internalization by MG63 cells in the absence of antibody was
defined as 100% internalization. C, The uptake of collagen in macrophages is independent of beta1-integrins.
The internalization of radiolabeled collagen type I was measured in cultured human macrophages in the
absence of exogenous reagents (column 1), or in the presence of 10 g/ml anti-beta1 mAb 4B4 (column 2),
or 200 M cyclic RGD peptide (column 3). D, Internalized collagen is degraded by lysosomal cysteine
proteases. The amount of collagen type I internalized by human macrophages was measured in the absence
of protease inhibitor (column 1) or in the presence of 20 M E64d (column 2). Internalization by untreated
macrophages was defined as 100% internalization. E, Macrophages internalize collagen in a mannose
receptor-dependent process. Activated macrophages were isolated from the peritoneum of either MR-/- mice
or littermate wildtype (MR+/+) mice 72 hours after thioglycollate injection. The internalization of collagen
was measured for MR+/+(column 1 and 2) and MR-/- macrophages (column 3 and 4) in the absence
(columns 1 and 3) or presence (columns 2 and 4) of 10 g/ml of mAb 5f4. Internalization by MR+/+
macrophages in the absence of antibody was defined as 100% internalization. F, Positive control for
mannose receptor-mediated endocytosis. Murine MR+/+and littermate MR-/- macrophages were assayed for
their ability to internalize radiolabeled mannose-BSA. For A-F, all samples were analyzed in triplicates.
Error bars indicate standard deviations.


b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

17

Table 1
Species Origin Reference
Epithelial cell lines
LNCaP human prostate carcinoma (72)
MDA-MB-231 human breast adenocarcinoma (73)
CaCo-2 human colorectal adenocarcinoma (74)
HeLa human cervix carcinoma (75)
MCF-7 human breast adenocarcinoma (76)
HEK-293 human embryonic kidney (77)
Lewis Lung Carcinoma (LLC) murine lung carcinoma (78)
B16F10 murine skin melanoma (79)

Mesenchymal cell lines
HGF-1 human healthy gingiva (80)
MG63 human osteosarcoma (81)
MRC-5 human lung fibroblast (82)
HT1080 human fibrosarcoma (83)
RD human rhabdomyosarcoma (84)
NCTC 2472 murine sarcoma (85)
NIH/3T3 murine embryonic fibroblast (86)





b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

B) A)
Collagen I
internalization
u
P
A
R
A
P
+
/
+
0
20
40
60
80
100
120
Holo-transferrin
internalization
Collagen I internalization by murine fibroblasts
D)
C)
Collagen I internalization by human sarcoma cells
E)
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
u
P
A
R
A
P
+
/
+

+

m
A
b

5
f
4
u
P
A
R
A
P
-
/
-
u
P
A
R
A
P
-
/
-

+

m
A
b

5
f
4
u
P
A
R
A
P
+
/
+
u
P
A
R
A
P
+
/
+

+

m
A
b

5
f
4
0
20
40
60
80
100
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
0
.
0
1
0
.
0
3
0
.
1
0
.
313
1
0
3
0
1
0
0
-
U
-
/
-

f
i
b
r
o
b
l
a
s
t
s
1
0
0

u
g
/
m
l
c
o
n
t
r
o
l

m
A
b mAb 5f4
( g/ml) 0
.
0
1
0
.
0
3
0
.
1
0
.
313
1
0
3
0
1
0
0
-
1
0
0

g
/
m
l
c
o
n
t
r
o
l

m
A
b

Fig.1
250
150
100
75
50
37
25
20
Mr
u
P
A
R
A
P
+
/
+
u
P
A
R
A
P
-
/
-
u
P
A
R
A
P
+
/
+

h
e
a
r
t
u
P
A
R
A
P
-
/
-

h
e
a
r
t
u
P
A
R
A
P
+
/
+

l
u
n
g
u
P
A
R
A
P
-
/
-

l
u
n
g
fibroblasts tissue
18

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Fig.2
+ 10 g/ml 5f4
Internalization of fluorescent-labeled collagen IV
+


E
6
4
d
C D
u
P
A
R
A
P
+
/
+

f
i
b
r
o
b
l
a
s
t
s
M
G
6
3
no Ab
E F
Internalization of fluorescent-labeled gelatin
B A
n
o

m
A
b
0
.
0
1

u
g
/
m
l

5
f
4
0
.
1

u
g
/
m
l

5
f
4
1

u
g
/
m
l

5
f
4
1
0

u
g
/
m
l

5
f
4
1
0
0

u
g
/
m
l

5
f
4
u
P
A
R
A
P

-
/
-

%

o
f

u
n
t
r
e
a
t
e
d

f
i
b
r
o
b
l
a
s
t
s
0
20
40
60
80
100
120
G Internalization of radio-labeled collagen IV
19

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Fig.3
20
Fibroblasts
MG63
Control mAb mAb 5f4
Control mAb mAb 5f4
uPARAP
Loading
control
uPARAP
Loading
control
A B
C
uPARAP level
%

o
f

a
n
t
i
-
T
N
P

t
r
e
a
t
e
d

c
e
l
l
s
0
20
40
60
80
100
120
Control
mAb
MG63
mAb 5f4
Fibroblasts
uPARAP level
%

o
f

a
n
t
i
-
T
N
P

t
r
e
a
t
e
d

c
e
l
l
s
0
20
40
60
80
100
120
Control
mAb
mAb 5f4
D

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

250
150
100
75
50
37
25
20
kDa
15
3/4 fragment
1/4 fragment
Intact -chains
1
:

I
n
t
a
c
t

c
o
l
l
a
g
e
n

I
2
:

C
l
e
a
v
e
d

c
o
l
l
a
g
e
n

I
3
:

u
P
A
R
A
P

+
/
+

f
i
b
r
o
b
l
a
s
t
s
4
:

u
P
A
R
A
P

-
/
-

f
i
b
r
o
b
l
a
s
t
s
5
:

u
P
A
R
A
P

+
/
+

f
i
b
r
o
b
l
a
s
t
s

+

5
0

g
/
m
l

5
f
4
B)
A)
Fig.4
21

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

uPARAP level
(% of reference cells)
0 20 40 60 80 100 120 140 160
C
o
l
l
a
g
e
n

i
n
t
e
r
n
a
l
i
z
a
t
i
o
n
(
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
)
0
20
40
60
80
100
120
140
A)
B)
uPARAP level
(% of reference cells)
0 20 40 60 80 100 120 140 160 180
0
100
200
300
400
500
600
(
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
)
H
o
l
o
-
t
r
a
n
s
f
e
r
i
n

i
n
t
e
r
n
a
l
i
z
a
t
i
o
n
Fig.5
22

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Collagen I internalization
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
uPARAP level
0
20
40
60
80
100
120
140
no antibody
mAb 5f4
u
P
A
R
A
P
+
/
+

f
i
b
r
o
b
l
a
s
t
s
H
G
F
-
1
M
R
C
-
5
M
G
6
3
N
C
T
C

2
4
7
2
N
I
H
/
3
T
3
H
T
1
0
8
0
H
e
L
a
L
L
C
M
D
A
-
M
B
-
2
3
1
R
D

c
e
l
l
s
B
1
6
F
1
0
H
E
K
-
2
9
3
M
C
F
-
7
L
N
C
a
P
C
a
C
o
-
2
u
P
A
R
A
P
-
/
-

f
i
b
r
o
b
l
a
s
t
s
Fig.6
23

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Collagen I internalization
0
20
40
60
80
100
120
140
160
180
uPARAP level
0
50
100
150
200
Holo-transferrin
internalization
0
20
40
60
80
100
A) B) C)
D)
Collagen I internalization
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
0
20
40
60
80
100
120
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
G
D

2
5
G
D

2
5
G
D

2
5
G
d
2
5

+

5
f
4
G
D
2
5
-
b
e
t
a
G
D
2
5
-
b
e
t
a
G
D
2
5
-
b
e
t
a
G
D
2
5
-
b
e
t
a

+

5
f
4
M
G
6
3
M
G
6
3

+

1
0

g
/
m
l

5
f
4
M
G
6
3

+

2

g
/
m
l

4
B
4
M
G
6
3

+

1
0

g
/
m
l

4
B
4
M
G
6
3

+

5
0

M

R
G
D
M
G
6
3

+

2
0
0

M

R
G
D
Fig.7
24

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Mannose-BSA internalization
0
20
40
60
80
100
120
140
Collagen I internalization
0
20
40
60
80
100
120
140
160
uPARAP level
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
0
20
40
60
80
100
120
A) B) C)
D) E) F)
%

o
f

r
e
f
e
r
e
n
c
e

c
e
l
l
s
M
R
+
/
+
M
R
-
/
-
Collagen I internalization
M
R
+
/
+
%

o
f

u
n
t
r
e
a
t
e
d

M
R
+
/
+

m
a
c
r
o
p
h
a
g
e
s
0
20
40
60
80
100
120
M
R
-
/
-
M
R
+
/
+
+
m
A
b

5
f
4
M
R
-
/
-
+
m
A
b

5
f
4
%

o
f

u
n
t
r
e
a
t
e
d

M
R
+
/
+

m
a
c
r
o
p
h
a
g
e
s
M
G
6
3
M
G
6
3
M
G
6
3
+
m
A
b

5
f
4
M
a
c
r
o
p
h
a
g
e
s
M
a
c
r
o
p
h
a
g
e
s
Collagen I internalization
0
100
200
300
400
500
%

o
f

u
n
t
r
e
a
t
e
d

m
a
c
r
o
p
h
a
g
e
s
M
a
c
r
o
p
h
a
g
e
s
M
a
c
r
o
p
h
a
g
e
s
+
E
6
4
d
M
a
c
r
o
p
h
a
g
e
s
+
m
A
b

5
f
4
Fig.8
Collagen I internalization
m
a
c
r
o
p
h
a
g
e
s
m
a
c
r
o
p
h
a
g
e
s

+

4
B
4
m
a
c
r
o
p
h
a
g
e
s

+

R
G
D
0
20
40
60
80
100
120
140
%

o
f

u
n
t
r
e
a
t
e
d

m
a
c
r
o
p
h
a
g
e
s
25

b
y

g
u
e
s
t

o
n

D
e
c
e
m
b
e
r

5
,

2
0
1
3
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m