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J. Biol. Chem., Vol. 275, Issue 45, 35432-35441, November 10, 2000
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From the Medical Research Council Group in Periodontal Physiology,
the
Received for publication, April 14, 2000, and in revised form, July 12, 2000
Intracellular collagen degradation by fibroblasts
is an important but poorly understood pathway for the physiological
remodeling of mature connective tissues. The objective of this study
was to determine whether gingival fibroblasts that express endogenous Phagocytosis is central to the uptake and degradation of
microorganisms as well as damaged or senescent cells and is therefore an essential process in host defense, tissue remodeling, and
inflammation. Although "professional" phagocytic cells such as
macrophages and neutrophils have been studied in considerable depth
(1), several types of nonphagocytic cells such as epithelial cells and
fibroblasts can internalize particles and matrix proteins in
vivo and in culture. Collagen fibril phagocytosis is thought to be
an important pathway for physiological degradation of extracellular
matrix in mature connective tissues (2). Uterine, wounded dermal, and
periodontal connective tissues exhibit rapid physiological turnover of
matrix proteins, processes that are mediated by the intracellular
degradation pathway (3). While in inflamed periodontal sites,
extracellular matrix metalloproteinases are believed to be responsible
for the bulk degradation of connective tissues (4), in normal turnover, fibroblasts are thought to use solely the intracellular vacuolar system
for focal proteolysis of collagen (3, 5). However, at present, the
vacuolar system that mediates intracellular collagen degradation is
incompletely characterized.
In professional phagocytes, the phagocytic process is initiated by
binding of particles to receptors on the plasma membrane, an event that
subsequently generates a phagocytic signal (6). In fibroblasts, the
initial internalization of intracellular collagen degradation is a
specific process that is mediated by the adhesive interactions between
ligand and collagen receptors (i.e.
Following particle internalization in macrophages, the resulting
intracellular vacuole (the phagosome) is subsequently transformed into
an acidic, hydrolase-rich phagolysosome. The transition from early
phagosome to phagolysosome involves the sequential fusion of endosomes
and lysosomes with phagosomes (9-11). While there are extensive data
on the phagocytic process and the regulation of phagosome
formation/maturation in macrophages (6), similar studies in the context
of intracellular collagen degradation in fibroblasts are limited.
Electron microscopic studies on the digestion of phagocytosed exogenous
collagen have shown cross-banded collagen profiles in the
phagolysosomes of fibroblasts (12, 13), and there is an apparent
requirement for lysosomal serine proteinases for collagen degradation
(13, 14). A confounding factor in fibroblasts is that not all nascent
collagen is secreted (15-17); a significant fraction is degraded
intracellularly in the endoplasmic reticulum, in the Golgi network, and
in the endosome/lysosome system, thereby complicating the distinction
between exogenous (i.e. phagocytosed) and newly synthesized
endogenous collagen.
To begin to address these questions, we have developed, characterized,
and validated a model system that uses periodontal fibroblasts in which
exogenous collagen binding is dependent on Reagents--
Paramagnetic, carboxylated (1-µm diameter) beads
were purchased from Bangs Laboratories Inc. (Fishers, IN). Antibodies
to gelsolin (clone GS-2C4), Bead Preparations--
Biotinylated collagen was prepared by
dissolving 10 mg of lyophilized porcine type 1 collagen in 0.01% HCl
overnight in the cold with constant stirring. Thereafter, the pH was
adjusted to 8.5. Sulfosuccinimidyl 6-(biotinamido)hexanoate
(Pierce; 2 mg/ml) was added to the collagen in two stages and incubated
for 20 min at each stage with constant stirring at 4 °C.
Biotinylated collagen was coupled to carboxylated magnetic
1-µm-diameter beads using a water-soluble carbodiimidine
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimidine hydrochloride. Beads
were washed with MES buffer (pH 6.0, 50 mM) three times at
room temperature. 1-Ethyl-3-(3-dimethylaminopropyl)hydrochloride (10 mg) was added slowly after bead sonication, and the mixture was
incubated for 30 min at room temperature and subsequently washed in
phosphate buffer (pH 8.2) before the addition of biotinylated collagen.
The reaction was stopped by incubating for 30 min with 10 mM ethanoalamine. Washed beads were stored in
phosphate-buffered saline containing 0.1% BSA and
NaN3.
Cell Culture--
Human gingival fibroblasts were obtained from
biopsies of normal gingiva in patients aged between 10 and 16 years as
described (10). These cells constitutively express abundant
Bead Incubation and Phagosome Isolation--
Cells were cooled
to 4 °C for 10 min before and after the addition of beads (cell/bead
ratio = 1:10) to allow bead binding but prevent internalization.
Cells were warmed to 37 °C to allow phagocytosis and were collected
at different time points. Cells were washed three times with
phosphate-buffered saline to remove any excess unbound beads.
Collagen-coated magnetic bead phagosomes were isolated by cell lysis in
homogenization buffer (1% Triton X-100, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5 mM EGTA, 20 µg/ml aprotonin, 1 µg/ml Pefabloc, and 10 mM Pipes (pH 6.8) by passage through a Dounce homogenizer
(30 times) over ice and washed (four times) after magnetic selection in
a Dynal MPC apparatus.
Fusion Assay--
Fusion of phagosomes with endosomes/lysosomes
was assessed by the addition of HRP-streptavidin (120 mg,
106 cells) to cells for 30 min followed by a 1-h chase in
Acidification of Phagosomes and Collagen Degradation--
Since
fluorescence of FITC is sensitive to pH, this dye was used to monitor
the acidity of intracellular vesicles (20). First, we examined the FITC
excitation spectra due to changes in the pH surrounding
fluorescein-streptavidin conjugated to biotinylated collagen-coated
beads. Calibration of the fluorescence ratio versus pH was
performed for each experiment by equilibrating the cells in the
presence of the K+/H+ ionophore nigericin (5 µM) at varying pH values. Calibration curves were
constructed by plotting the extracellular pH (assumed to be identical
to the cytosolic pH under these conditions) against the corresponding
fluorescence ratio. We validated the ability of A7 antibody to
recognize degraded collagen (21) by incubating native collagen-coated
beads with purified cathepsin B in the presence of 1 mM
EDTA and 1 mM cysteine for 4 h in phosphate buffer, pH
5.5, at 37 °C. The amount of protein on beads was determined by a
Bio-Rad assay. Cathepsin B-treated and native collagen-coated beads
were incubated with A-7 antibody followed by secondary antibody (FITC-goat anti-mouse antibody). Beads were assessed by fluorescence microscopy or flow cytometry.
Electron Microscopy and Flow Cytometry--
Bead internalization
was conducted as described above, and fibroblasts were fixed in 1%
glutaraldehyde for 1 h. Samples were embedded in Epon 812, and
thin sections were placed on nickel grids. Sections were stained with
uranyl acetate and lead citrate and observed under an electron
microscope (Hitachi-60). Counts of immunogold labeling for LAMP-2 or
background counts of immunogold particles were obtained from samples
incubated with secondary antibody only or with an irrelevant isotype control.
Bead internalization and intracellular degradation of the collagen
bound to phagocytosed beads were studied after fixation, staining with
A7 at pH 7.4, and analysis by flow cytometry. Collagen and BSA
internalization and surface Calcium Measurement--
To examine the role of intracellular
calcium in phagocytosis, cells were loaded with fura2/AM (3 µM), and ratio fluorimetry of single cells was used to
estimate intracellular calcium ion concentration
([Ca2+]i) as described (22).
Human Gingival Fibroblasts Phagocytose Collagen-coated Beads
through the
Phagosomal compartments containing the paramagnetic particles were
recovered from whole cell homogenates using a magnet (23). This
procedure yielded pure preparations of phagosomes and showed little
contamination by cell debris or by unbroken whole cells (Fig.
1B). Immunoblotting of bead-associated proteins at 30 min showed no significant lysosome-associated protein (LAMP-2; Fig. 2A), a marker of late
endosomes and lysosomes that appeared only at later times following
bead incubation. Thus, the phagosome preparations were free of
lysosomal membrane contamination at the outset of the experiments.
The association of plasma membrane proteins with beads prepared by
magnetic pull-offs was examined by immunoblotting with an anti-annexin
antibody. There was an initially high level of annexin that decreased
to nearly undetectable levels by 30 min (Fig. 1C). This
observation suggested an early engagement of surface membrane with the
particle surface as the pseudopod enveloped the bead, a contention that
was consistent with electron microscopic studies at initial binding
times demonstrating pseudopod extensions around the beads (Fig.
1D). In gingival fibroblasts, the
Protein Composition of Phagosomes during
Maturation--
Immunoblot analysis of phagosomes at various stages
after bead binding showed an initial increase in the amount of
Immunoblotting of bead preparations for cathepsin B showed a
progressive increase between 30 and 240 min, while the level of LAMP-2
increased from 60 to 240 min. Thus, the enzymes involved in collagen
degradation start to appear in the phagosomes in parallel with the
maturation of phagosomes to phagolysosomes. Quantitative analysis of
LAMP-2 localization around the periphery of beads by immunoelectron
microscopy demonstrated an increase from 26 gold particles/100
µm2 after 10 min to 290 gold particles/100
µm2 after 120 min. Background counts of gold particles
per 100 µm2 (no primary antibody) were subtracted in both
cases, and these background counts with no primary antibody were very
similar to those obtained with an irrelevant isotype control antibody.
Fusion of Phagosomes with Endosomes/Lysosomes--
We assessed
vacuolar membrane proteins associated with internalized collagen-coated
beads. Cells were incubated with soluble FITC-streptavidin, washed, and
chased followed by incubation with biotinylated-collagen beads. This
assay distinguished internalized beads from external beads,
since only internalized beads exhibited FITC fluorescence. We used
LDL-DiI and LAMP-2 immunostaining to study the spatial association of
endosomes with lysosomes. For study of endosome-phagosome fusion, cells
were loaded with FITC-streptavidin and biotinylated collagen-coated
beads, followed by labeling cells with the endosomal marker LDL-DiI.
Fluorescent endosomal vesicles were readily visualized with LDL-DiI
(Fig. 3A, a).
Transmitted light images (Fig. 3A, e) showed both
external beads (solid arrows) and internalized
beads (open arrows); the latter were
differentiated from external beads by FITC fluorescence (Fig.
3A, f). For samples obtained 30-240 min after
bead incubation, we determined the number of internalized beads that
co-localized (yellow fluorescence) with LDL-DiI
(Fig. 4) in small vesicular bodies
resembling endosomes (Fig. 3A, b-d, 30 min after
labeling; e-h, 90 min after labeling). At 30 min, there was
45% co-localization of internalized collagen-coated beads with
LDL-DiI, which decreased to nearly 0% at 240 min (Fig. 4).
Immunostaining for LAMP-2 showed punctate staining in untreated cells
(Fig. 3B, a). The spatial distribution of
internalized beads (Fig. 3B, b) colocalizing with
LAMP-2 immunostaining (Fig. 3B, d) also permitted
quantitative analysis of LAMP-2-phagosome association (Fig. 4). There
was a progressive increase in the spatial association of LAMP-2 with
phagosomes from 30 to 240 min.
We distinguished surface-bound from internalized probe with an
anti-FITC antibody to quench extracellular FITC fluorescence. The
quenching efficiency of anti-FITC antibody was determined by adding
antibody to FITC-streptavidin-loaded cells followed by cell
permeabilization and fluorimetric quantification. The mean fluorescence
intensity of FITC-streptavidin-loaded cells was 37.2 ± 3.6 (n = 10 cells) and 36.1 ± 4.2 (n = 10 cells) after the addition of anti-FITC quenching antibody to
unpermeabilized cells. In contrast, the mean fluorescence intensity of
permeabilized cells decreased to 15.5 ± 2.3 units
(n = 10 cells) after the addition of anti-FITC
quenching antibody, indicating that the FITC-streptavidin was indeed
internalized. We assayed the extracellular medium from fusion assays
with biotin-HRP and found that the amount of exocytosed probe was
negligible. Similarly, we studied FITC-streptavidin fluorescence at
different time points (0-240 min) and found that lysosomal enzymes did
not reduce FITC fluorescence.
Biochemical analysis of the fusion product of endocytosed
HRP-streptavidin and phagocytosed biotinylated collagen-coated beads also showed increasing amounts of fusion over 30-240 min (Fig. 5). Since endosome/lysosome trafficking
is thought to require cycles of assembly and disassembly of
microtubules and actin (25, 26), phagosomal maturation in fibroblasts
may also require the coordinated interaction of the actin-based and
tubulin-based cytoskeletons. Accordingly, in samples treated with
cytochalasin D (1 µM) or colchicine (10 µM), there was a nearly complete block of fusion with
cytochalasin and a 73% reduction with colchicine between 30 and 240 min (Fig. 5).
Collagen Degradation during Phagosomal Maturation--
We
determined whether there was progressive degradation of the native
collagen coating the beads with an antibody (A-7) that specifically
recognizes degraded (but not native) collagen. To verify that the A-7
antibody recognizes degraded collagen, native collagen-coated beads
were treated with cathepsin B (0.5 units) over varying times in the
presence of 1 mM EDTA and 1 mM cysteine (in
phosphate buffer, pH 5.5, at 37 °C). The beads were fixed and
immunostained with A-7 antibody, and the fluorescence intensity was
measured with a cooled CCD camera (Pentamax; Princeton Instruments) interfaced to a microscope. There was a gradual increase in
fluorescence intensity over time with cathepsin B treatment (Fig.
6A). Since cathepsin B
degrades collagen by an initial cleavage of telopeptides to release
fibrils (27, 28), our results suggest that the A-7 antibody (21)
recognizes an epitope exposed during the initial unfolding of collagen
by cathepsin B. Accordingly, we used the A-7 antibody and flow
cytometry to examine during phagocytosis the temporal degradation of
native collagen coated on the beads. There was a steady increase in the
amount of degraded collagen up to 120 min that decreased slightly at
240 min, possibly due to proteolytic epitope degradation (Fig.
6B).
Since nascent collagen can also be degraded intracellularly (15-17),
we used a higher resolution confocal microscopy assay that measures
only bead-associated collagen degradation in the cells and thus
discriminates spatially from potential intracellular nascent collagen
degradation elsewhere. Fluorescent collagen-coated beads were incubated
with cells for different times and then sorted by flow cytometry on the
basis of cells with and without beads. The collected cells were fixed
and sedimented to slides using cytocentrifugation, and the presence of
degraded collagen associated solely with beads was determined with the
A-7 antibody and confocal microscopy. There was a nearly 2-fold
increase in bead-associated fluorescence between 10 and 120 min after
bead internalization of native collagen-coated beads (Table
I).
Biochemical analysis of bead-associated collagen in the phagosomes was
detected on blots by probing with HRP-streptavidin followed by
development with ECL reagents. We confirmed the integrity of the triple
helical structure of biotinylated collagen (0.5 mg/ml) used in our
studies by treating with trypsin (0.05 mg/ml; pH 7.5 for 18 h at
37 °C; Fig. 6C, a). Following incubation with cells, the biotinylated collagen attached to the beads showed degradation into smaller fragments, which appear below the Phagosome Acidification Is Required for Collagen Degradation but
Not Phagosomal Maturation--
Since cathepsin B requires an acidic
phagosomal compartment (28, 29) to effect collagen degradation (27), we
examined the pH of the bead-associated compartment. To calibrate the
intracellular response of FITC-streptavidin-biotinylated collagen beads
to changes in pH, we used nigericin-KCl (30), which enabled us to
equilibrate the pH in the cell with that of the medium (Fig.
7A). The pH dependence of FITC
fluorescence (20) allowed us to measure the intraphagosomal pH of
bead-associated collagen. We established a calibration curve that
showed the relationship between excitation fluorescence ratios of
440/490 nm and the pH of the solution surrounding the collagen bead
(Fig. 7A). Since not all of the particles that bind to cells become internalized, we verified the intracellular location of beads by
abruptly acidifying the extracellular pH (pH 6). This intervention
greatly reduced the fluorescence ratio of FITC-labeled extracellular
particles (pH 6.0) but had little effect on the fluorescence ratio of
intracellular and presumably intraphagosomal particles, which indicated
a relatively constant pH, ~5.4-5.6. Conversely, after the addition
of NH4Cl to the medium, the extracellular pH was hardly
affected (pH 7.45), while the intracellular bead-associated pH
increased to pH 7.1. Of the 50 presumptive phagosomal particles that
were studied in these experiments, 45 (90%) were confirmed to be in an
intracellular compartment by this method. Results shown in Fig.
7B were determined from beads shown to be internalized. After the addition of labeled beads, the pH of the compartment (phagosome) containing the collagen bead decreased from 7.4 to 5.4 over
120 min, and by 240 min the mean pH had reached 5.6 (Fig. 7B).
We used concanamycin A to prevent vacuolar acidification. From initial
experiments with concanamycin A and acridine orange (1 µg/ml), we
determined the optimal concentrations and time of incubations required
(31). Because of the high protein/volume ratio in the assays, we used
10 µM concanamycin A for 1 h to obtain complete
inhibition of phagosomal acidification. After concanamycin treatment,
the pH for FITC-labeled phagolysosomes ranged from 7.13 to 7.09 for up
to 240 min of incubation (Fig. 7B). The ability of
concanamycin to inhibit intralysosomal acidification was also estimated
by measuring the fluorescence emission of endocytosed FITC-streptavidin. Incubation of fibroblasts with FITC-streptavidin for
60 min resulted in accumulation of fluorescent probe within the cells
as a faint, punctate lysosome-like pattern (Fig. 7C, left). Treatment with 10 µM concanamycin A
followed by incubation with FITC-streptavidin increased the
fluorescence intensity with 490-nm excitation (Fig. 7C,
right), indicating that the pH of the fluorescent
compartments had increased. Notably, treatment with concanamycin A did
not affect the fluorescence of FITC-labeled extracellular beads.
We determined if acidification is required for intracellular collagen
degradation. Cells were preincubated for 1 h with concanamycin A
followed by collagen bead internalization. After fixing,
permeabilization, and staining with the A-7 antibody at pH 7.4 to label
degraded but not native collagen, we found greatly reduced fluorescence at intracellular bead sites in the concanamycin-treated cells (Fig.
8, A and B). In
comparison, vehicle-treated cells (Me2SO only) exhibited
bright fluorescence staining (Fig. 8, C and D). These findings indicated that inhibition of acidification prevents degradation of collagen. This notion was also confirmed by analyzing bead-associated protein after 240 min in the presence of concanamycin (Fig. 6C). Lane b shows undegraded
To determine if phagosomal maturation is affected by treatment with
concanamycin, we performed phagocytosis assays with FITC-streptavidin and biotinylated collagen beads as described for the fusion assay above. After 240 min of chase, fixed and permeabilized cells were immunostained with cathepsin B antibody. Fig.
9A (c) shows close spatial colocalization of cathepsin B with beads in cells treated with
concanamycin. Further, treatment with concanamycin did not effect the
ability of cells to internalize beads; there was 24 ± 2%
collagen bead phagocytosis in untreated cells compared with 23 ± 3% in concanamycin-treated cells (1 h pretreatment; 60-min bead
incubation). Similarly, fusion of phagosomes with endosomes/lysosomes was not affected by concanamycin when used in experiments as described in Fig. 5 (Fig. 9B), suggesting that dissipation of the pH
gradient had no effect on the fusion process. Further, treatment with
concanamycin did not affect the association of LAMP-2 or the vacuolar
enzyme cathepsin B with phagosomes (Fig. 9C).
Role of Calcium in Phagosomal Acidification--
We assessed the
requirement for calcium in phagosome formation and maturation (32). The
addition of collagen beads to cells induced a robust increase of
[Ca2+]i (Fig.
10A). Depletion of
intracellular stores with thapsigargin (5 µM) reduced the
[Ca2+]i response to the beads (Fig.
10B). Similar results were seen by preloading cells with the
intracellular calcium-chelating agent BAPTA/AM (3 µM;
Fig. 10C). In parallel experiments to assess regulation of
phagosomal acidification, intracellular calcium store depletion or
chelation of intracellular Ca2+ prevented phagosomal
acidification (Fig. 10D). Jointly, these experiments
indicate that We have developed, characterized, and validated a model system in
which biotinylated collagen-coated magnetic beads were used to perform
quantitative biochemical analyses of bead-associated proteins on
relatively pure populations of phagosomes at different stages of
formation and maturation. A major advantage of the new model system is
that the bead-bound biotinylated collagen permits simultaneous study of
fusion processes and intracellular degradation of exogenous
collagen in fibroblasts. This system enables clear cut discrimination
of the exogenous collagen from nascent collagen (15) that also
undergoes intracellular degradation (16).
Currently there are no data on the protein composition of maturing
phagosomes or on the regulation of the lysosomal maturation process
that leads to intracellular collagen degradation. Our experiments were
conducted so that initial bead binding at 4 °C was followed by
subsequent warming to 37 °C, an approach that enhanced the entry of
a cohort of internalized beads into the intracellular trafficking
pathways. Data from these experiments demonstrated that high levels of
collagen receptors and the cell membrane protein annexin were
associated with phagocytosed beads at early time points and declined
shortly thereafter. These data, the antibody-blocking experiments, and
previous findings (7, 8, 19) show that the collagen protein associated
with the collagen-coated beads in parallel with actin. This association was contemporaneous with the initial rearrangement of the cell membrane
in collagen bead phagocytosis, a process that involved extension and
wrapping of cell processes around the bead. The initial bead
internalization process is thought to require rearrangements of
cortical actin filaments (24), a contention that is supported by
Desjardins et al. (11), who showed temporal variations of the amounts of actin and actin-binding proteins associated with maturing phagosomes in macrophages.
The presence of LAMP-2 (a late endocytic and lysosomal marker) in
phagosomes was negligible at early stages of bead internalization but
quickly increased to maximal levels from 60 to 240 min. These results
are similar to previous observations in macrophages (9, 34) showing
that LAMP-1 increases in abundance from the transition of endosomes to
lysosomes. Notably, the levels of bead-associated cathepsin B increased
slowly from 30 to 240 min as would be expected if collagen degradation
were to be effected in vacuolar compartments.
Fusion and Collagen Degradation--
As assessed by
bead-associated HRP activity or FITC fluorescence, vesicle fusion was
increased from 30 to 240 min. These data indicate that when phagosomes
form, they begin fusing with early endosomes. In the endosome
maturation model (35, 36), early endosome-endosome exchange is proposed
to lead to formation of lysosomal compartments. Similarly in
fibroblasts, fusion of phagosomes appears to occur at later stages of
endosome maturation. Indeed, maximal fusion between phagosomes and
lysosomes occurred after 120 min of bead internalization, since the
lysosomal protein LAMP-2 was most abundant on beads from 60 to 240 min.
In fibroblasts, the digestion of internalized fibrillar collagen
depends to a large extent on the activity of cysteine proteinases (e.g. cathepsin B; Ref. 3). Our data demonstrate
association of cathepsin B with the collagen bead-containing phagosomes
starting at 30 min and steadily increasing up to a maximum at 240 min. Since the exo- and endopeptidase activities of cathepsin B are dependent on an optimal pH (27), we measured the pH of the collagen bead compartment. Our data illustrate that there is a substantial increase in the hydrogen ion concentration in collagen bead-containing phagolysosomes over time. We used concanamycin A, a relatively specific
inhibitor of the V-ATPase (31), to reduce formation of a pH gradient.
In our system, inhibiting acidification reduced collagen degradation
but did not affect fusion events leading to phagosomal maturation. This
is in contrast to results showing that the V-ATPase inhibitor
bafilomycin A1 affects transit from early to late endosomes
(39) and late endosomes to lysosomes (40) and suggests that phagosomal
maturation is a distinct pathway. Experiments in which depletion of
intracellular calcium stores reduced phagosomal acidification in
fibroblasts indicate that processes that lead to acidification of
phagosomes are, at least in part, calcium-dependent. In
this context, there have been conflicting reports on the requirement of
calcium transients in neutrophils (41) and macrophages (42) for
phagocytic function.
Perturbation of intracellular digestion of collagen is crucial in the
pathogenesis of a wide variety of drug-induced fibrotic lesions of
connective tissues (42). To characterize the intracellular collagen
degradation pathway, we utilized cells from periodontal tissues, since
they are known to be avidly phagocytic and digest collagen via the
intracellular route (2, 3, 5). The use of these cells and of
biotinylated collagen-coated magnetic beads has enabled detailed
characterization of an We thank Sergio Grinstein for advice.
*
This work was supported by the Canadian Institutes of Health
Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Rm. 244, Fitzgerald Bldg., University of Toronto, 150 College St., Toronto,
Ontario M5S 3E8, Canada. Tel.: 416-978-6684; Fax: 416-978-5956; E-mail: christopher.mcculloch@utoronto.ca.
Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M003221200
The abbreviations used are:
FITC, fluorescein
isothiocyanate;
HRP, horseradish peroxidase;
LDL, low density
lipoprotein;
DiI, 1,1'-dioctadocyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate;
BSA, bovine serum albumin;
MEM, minimal essential medium;
MES, 4-morpholineethanesulfonic acid;
Pipes, 1,4-piperazinediethanesulfonic
acid;
BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid/acetoxymethyl ester.
A Novel Model System for Characterization of Phagosomal
Maturation, Acidification, and Intracellular Collagen Degradation
in Fibroblasts*
, Faculty of Dentistry, and the § Faculty of
Medicine, Division of Respirology, University of Toronto, Toronto M5S
3E8, Ontario, Canada and the University Health Network Research
Institute, Toronto M5G 2C4, Ontario, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 integrin, the collagen receptor,
would exhibit the cellular machinery required for phagosomal maturation
and collagen degradation. There was a time-dependent
increase of collagen bead internalization and a
time-dependent decrease of bead-associated
21 integrin after initial bead binding.
-Actin and gelsolin associated transiently with beads (0-30 min)
followed by LAMP-2 (60-240 min) and cathepsin B (30-240 min).
Cytochalasin D prevented phagosome formation and also prevented the
sequential fusion of early endosomes with lysosomes. Collagen
bead-associated pH was progressively reduced from 7.25 to 5.4, which
was contemporaneous with progressive increases in degradation of
bead-associated collagen (30-120 min). Concanamycin blocked
acidification of phagolysosomes and collagen degradation but not
phagosome maturation. Phagosomal acidification was partly dependent on
elevated intracellular calcium. These studies demonstrate that the
cellular machinery required for intracellular collagen degradation in
fibroblasts closely resembles the vacuolar system in macrophages.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21 integrins; Ref. 7). However, little is
known about the regulation of the downstream events and the kinetics of
the intracellular phagocytic pathway following the initial binding of
collagen to the cell surface (8).
21 integrin expression (7). Biotinylated
collagen-coated magnetic beads were used to perform quantitative
biochemical analyses of bead-associated proteins on relatively pure
populations of phagosomes at different stages of formation/maturation.
A major advantage of the new model system is that the bead-bound
biotinylated collagen permits study of fusion processes and
intracellular degradation of exogenous collagen in fibroblasts and
simultaneously enables clear cut discrimination from nascent collagen
that is undergoing degradation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (clone AC-15), bovine type 1 collagen (clone COL-1), cytochalasin D, colchicine,
4'-6'-diamidino-2-phenylindole, fluorescein isothiocyanate
(FITC)1-conjugated goat
anti-mouse antibody, cathepsin B, trypsin, and tetramethyl
rhodamine isothiocyanate-phalloidin were from Sigma. Antibody to
degraded collagen was provided by Osteometer (A7 clone; Denmark).
Rabbit polyclonal antibody to human cathepsin B was from Vital Products
Inc. (St Louis, MO). Antibody to LAMP-2 (clone H4B4) was from
Developmental Studies Hybridoma Bank (Iowa City, IA). Mouse monoclonal
anti-human integrin antibodies were obtained to the 1
subunit (clone A431 (Transduction Laboratories), clone 4B4 (Coulter))
and to the 2 subunit (clone P1E6 (Calbiochem), clone 3S3
(Serotec)). Horseradish peroxidase (HRP)-streptavidin, antibody to
FITC, low density
lipoprotein-1,1'-dioctadocyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (LDL-DiI), nigericin, and acridine orange were from Molecular Probes, Inc. (Eugene, OR). FITC-labeled streptavidin was from
Roche Molecular Biochemicals. ImmunoPure sulfosuccinimidyl 6-(biotinamido)hexanoate and water-soluble carbodiimidine
(1-ethyl-3-(3-dimethylaminopropyl) hydrochloride) were from Biolynx
(Brockville, Ontario, Canada; Pierce).
21 (18), and phagocytosis of exogenous
collagen requires this integrin (7, 19). Cells between passages 3 and
12 were used for all experiments.
-MEM after washing four times in -MEM (no serum). Biotinylated
collagen beads were added to cells and incubated for 30, 60, 120, and
240 min. The phagosomes were prepared as described above and
dot-blotted onto nitrocellulose paper, and HRP enzymatic activity due
to HRP-streptavidin-biotinylated collagen interaction was developed
with ECL reagents and quantified by densitometry. To study localization
of collagen-coated beads with respect to phagosomes and
endosomes/lysosomes, FITC-streptavidin (40 µg/ml) was used instead of
HRP-streptavidin as described above.
21 staining
were examined as described (7).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21 Integrin--
Human
gingival fibroblasts were presented with collagen-coated beads, and the
temporal relationship between bead binding and bead internalization was
studied by conducting bead binding at 4 °C with subsequent warming
to 37 °C for synchronization. To differentiate between internalized
and cell surface-bound beads, collagen-coated beads were incubated with
cells and were later stained with antibodies for immunofluorescence
localization of collagen. Since cells were neither fixed or
permeabilized, viable cells with intact cell membranes excluded
antibody staining of collagen beads. This approach used microscopy to
distinguish surface-bound (fluorescent) beads from internalized
nonfluorescent beads. With these methods, we found a progressive
increase in the number of internalized beads per cell over 30 min of
incubation (Fig. 1A).
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Fig. 1.
Bead internalization. A, to
distinguish internalized from cell surface-bound beads, collagen-coated
beads were incubated with unfixed cells followed by immunostaining for
collagen with anti-collagen antibody and FITC-conjugated secondary
antibody. Surface-bound (i.e. noninternalized) beads
exhibited green fluorescence, while internalized beads were excluded
from the antibodies and were nonfluorescent. Total bead counts and
FITC-fluorescent bead counts were made by microscopy. The number of
non-FITC fluorescent beads per cell (i.e. internalized
beads) increased from 0 to 30 min and stabilized between 30 and 60 min.
B, phagosomes were prepared as described under
"Experimental Procedures." Paramagnetic beads used for the assays
were synthesized from polystyrene microspheres infused with colloidal
oxide. Electron microscopy shows microspheres of polystyrene on the
periphery of individual beads. There were no unbroken cells and no
detectable cell debris in the phagosomal preparation. Note the uranyl
acetate-stained phagosomal proteins around the perimeter of the beads
(arrows). C, phagosome preparations at different
times after bead incubation were probed with annexin antibody. Note the
initially high annexin levels; these decrease during later stages of
phagocytosis. D, early step of phagocytosis in gingival
fibroblasts showing bead entrapment. Phagocytosing cells at 15 min were
fixed in 3% glutaraldehyde and observed by electron microscopy. Note
the cytoplasmic extensions around the bead (arrows). Latex
beads were used for this assay and therefore appear different from the
bead structure in B. E, 2 and
1 integrins detected by immunoblotting show initially
high levels followed by a decline thereafter, suggesting that after
internalization there is a loss (or recycling) of integrins from the
bead.
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Fig. 2.
Phagosome-associated proteins.
A, phagosomes were collected as described under
"Experimental Procedures," and bead-associated proteins were
assessed by immunoblotting. Note the transient increase in actin and
gelsolin (an actin-binding and -severing protein). Cathepsin B, an
enzyme involved in collagen degradation, starts to appear at 30 min and
increases up to 240 min, suggesting that phagosomes and lysosomes begin
to fuse at 30 min. Lysosome-associated membrane protein (LAMP-2) is
detected from 60 to 240 min. B, immunofluorescence staining
of 15.8-µm diameter latex beads shows co-localization of actin
(C) and gelsolin (D) around the bead periphery
at 10 min.
21 integrin is required for phagocytosis
of collagen-coated beads (7, 19). We studied the specificity of this
pathway with collagen and BSA-coated beads and with blocking antibodies
to the 21 integrin. After 1-h
incubations, there were >5-fold higher percentages of cells binding
collagen beads than BSA beads (collagen = 29.7 ± 1.5%;
BSA = 5.7 ± 0.3%). Pretreatment with blocking antibodies showed a 10-fold reduction of binding with collagen beads but only a
small reduction with BSA beads (collagen plus antibody = 2.9 ± 1.7%; p < 0.01; BSA plus antibody = 4.7 ± 0.4%; p > 0.1). Immunostaining for surface
2 by flow cytometry analyses showed a
time-dependent reduction after bead incubation
(t0 = 53 ± 3 fluorescence units;
t180 = 25 ± 6 fluorescence units).
Immunoblots of bead-associated 2 and 1
integrin subunits showed an initially high level that was reduced to
very low levels by 60 min, suggesting that after bead internalization
there was a loss (or recycling) of integrins from the bead (Fig.
1E). Collectively, these results indicate that
collagen-coated beads are phagocytosed by fibroblasts through the
21 integrin.
-actin (0-5 min) that declined thereafter by 60 min (Fig.
2A). These increased levels of -actin were accompanied by
a parallel increase in the amount of the actin-binding protein
gelsolin. For all analyses of bead-associated proteins, equal amounts
of beads were assessed at each time point. Immunolabeling of intact
cells with antibodies to -actin and gelsolin demonstrated that these
two proteins colocalized with internalized beads and formed a ring of
staining around the bead perimeter (Fig. 2B). Cytochalasin
D, a toxin that depolymerizes actin and reduces collagen phagocytosis
in vitro (8, 24), prevented the recruitment of actin and
gelsolin to beads (data not shown), consistent with an involvement of
actin and gelsolin in phagosome formation. We measured the percentage
of cells that bound collagen beads in the presence of cytochalasin D (1 µM). There was no significant difference between bead
binding in treated cells (23.9 ± 4.2%) and untreated cells
(24.8 ± 3.2%; p > 0.2).
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Fig. 3.
A, colocalization of the endosomal
marker (LDL) and internalized beads. a, cells not engaged in
phagocytosis were incubated with LDL-DiI (red) for 30 min,
chased for 10 min. b and f, cells exposed to
FITC-streptavidin for 30 min, chased for 10 min followed by incubation
with biotinylated collagen-coated beads for 30 and 90 min,
respectively. Only internalized beads appear green
because of fusion with FITC-streptavidin. e, transmitted
light microscopy (90 min) shows the locations of beads;
solid arrowheads show external beads, and
open arrowheads show internalized beads.
c and g, pattern of LDL staining in a phagocytic
cell (same as in b) after 30 and 90 min of chase,
respectively. d and h, colocalization of
phagocytosed internalized particles and LDL-DiI at 30 and 90 min
(yellow particles). B, colocalization
of internalized beads and LAMP-2. Cells were loaded with
FITC-streptavidin and biotinylated beads as described above, and
internalized beads were distinguished by the same criteria as in
A. Fixed and permeabilized cells were immunostained for
LAMP-2. Optical sections of cells by confocal microscopy show overall
distribution and co-localization of phagolysosomes labeled for LAMP-2
after a 240-min chase. a, pattern of LAMP-2 staining in
untreated fibroblasts. b, internalized beads. c,
LAMP-2 distribution in a phagocytic cell. d, yellow staining
shows discrete sites of co-localization of phagosomes with LAMP-2 in a
cell process.
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Fig. 4.
Association of endosomal and lysosomal
markers with phagosomes. Cells were incubated with LDL-DiI for 30 min, chased for 10 min with MEM, exposed to FITC-streptavidin for 30 min, and then chased for 10 min with MEM followed by incubation with
biotinylated collagen-coated beads. The cells were fixed at 30, 60, 120, and 240 min, and the percentage of beads that co-localized with
LDL-DiI was computed. To assess the temporal association of lysosomes
with phagosomes, cells were loaded with FITC-streptavidin and beads as
described above; fixed at 30, 60, 120, and 240 min; permeabilized; and
immunostained for LAMP-2. Internalized beads were identified by FITC
fluorescence on the beads as described in Fig. 3 (n = 50 cells for each group; data are means and S.E.).
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Fig. 5.
Biochemical fusion assay for endocytic and
phagocytic pathways. HRP-streptavidin (120 mg, 106
cells; endocytic probe) was added to cells for 30 min followed by
1 h of chase in MEM after washing four times in MEM (no serum).
Biotinylated collagen beads were added to cells and incubated for 30, 60, 120, and 240 min. Phagosomes/phagolysosomes were prepared as
described under "Experimental Procedures" and dot-blotted,
and peroxidase activity due to streptavidin-biotin interaction was
detected by ECL and quantitated by densitometry. As shown in control,
there is increasing fusion of the HRP-streptavidin with the
biotinylated collagen beads over time. Pretreatment and the continuous
presence of cytochalasin D (1 µM) during the experiment
reduce fusion to low levels between 30 and 240 min. To a lesser extent,
colchicine (10 µM) inhibited the fusion process.
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Fig. 6.
Degraded collagen detected by A-7
antibody. A, histogram of A-7 staining of native
collagen beads (control) or collagen beads incubated with
cathepsin B for the indicated times. Data are mean ± S.E. of
fluorescence intensity (n = 10 beads/sample).
B, flow cytometry analysis of A-7 staining showing temporal
degradation of collagen in fibroblasts. Phagocytosis assay was
performed with native collagen beads. Note the increase in degraded
collagen over time with a small decline at 240 min. Second antibody
only control used native collagen-coated beads stained without A-7
antibody (5 fluorescence units). Native beads without intracellular
digestion but stained with A-7 antibody exhibited 11 fluorescence units
(not shown). C, phagocytic assays were performed with
biotinylated collagen-coated beads. Bead-associated proteins were
separated by SDS-PAGE, and blots were probed with HRP-streptavidin to
reveal biotinylated collagen chains. Lane a shows
resistance of collagen to trypsin treatment for 18 h, pH 7.4, at
37 °C, indicating the triple helical structure of the native
collagen on the bead. Lane b shows undegraded
, , and 
collagen chains in phagosome preparations from cells
preincubated with concanamycin before bead incubation for 240 min. In
lanes c-f, cells were incubated with beads for
30, 60, 120, and 240 min, respectively. Note the lower molecular mass
collagen fragments and the smear, indicating progressive collagen
degradation over time.
Cells were incubated with native collagen or cathepsin B-degraded
collagen. UV beads were sorted by flow cytometry on the basis of UV
bead fluorescence. Beads were stained with A-7 antibody and examined by
confocal microscopy. Data are means ± S.E. of fluorescence
intensity units, derived by confocal microscopy of collagen
bead-associated fluorescence. n = 10 cells/group.
*, statistically significant difference compared with
control (p < 0.05). Note that different fluorescence
imaging systems were used to measure the beads in this table compared
with Fig. 6A.
chains, indicating that the collagen was indeed undergoing degradation in the
phagolysosome over time (Fig. 6C, lanes
c-f). These results are consistent with the collagen
degradation mediated by cathepsin B at pH 5.5 (measured with A-7
staining; Fig. 6A) and are similar to previous in
vitro studies showing the effect of pH on cathepsin-mediated collagen degradation (27).
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Fig. 7.
Phagosomal pH. A, calibration
of ratiometric fluorescence of FITC-streptavidin-biotinylated collagen
beads with changes in pH. Cells containing FITC-labeled phagolysosomes
were treated with nigericin-KCl, which enabled equilibration of
intracellular pH with that of the medium (n = 4).
A shows the results of one of the four experiments
performed. B, after 10 min of bead attachment at 4 °C,
cells plated on coverslips were transferred to 37 °C, and pH was
estimated by microscopic ratio spectrofluorimetry over time.
Internalized beads were distinguished from external beads by the
criteria described in Fig. 3. To determine if phagosomal acidification
is mediated by vacuolar ATPases, cells were pretreated with 10 µM concanamycin A for 1 h followed by incubation
with FITC-streptavidin-biotinylated collagen beads. (n = 5). C, fluorescence micrographs of cells after incubation
with soluble FITC-streptavidin for 4 h followed by incubation for
1 h with vehicle (left) or 10 µM
concanamycin (right). Microscopy shows fluorescent,
punctate, lysosome-like pattern with increased fluorescence intensity
in samples treated with concanamycin.
, , and collagen chains in phagosome preparations from cells
preincubated with concanamycin before bead incubation.
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Fig. 8.
Acidification and collagen degradation.
Concanamycin-treated cells (A and B) and
vehicle-treated cells (C and D) were incubated
with collagen-coated sulfated UV beads. After 120 min, cells were
immunostained with A-7 antibody to show degraded collagen on beads.
B and D show identical fields of A and
C. A and C show UV fluorescence of
beads and 4'-6'-diamidino-2-phenylindole-stained nuclei. The
circled beads in A and C
represent beads that were not internalized and therefore were unstained
by A-7. The arrowheads in D show internalized
beads that stained with A-7 antibody.
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Fig. 9.
Effect of concanamycin on bead
internalization and fusion process. A, cells were
pretreated for 1 h with 10 µM concanamycin
(conA), incubated with collagen beads for 2 h, fixed,
permeabilized, and immunostained for cathepsin B. a,
FITC-streptavidin staining of biotinylated beads;
b, cathepsin Bimmunostained cells; c,
co-localization of FITC-streptavidin and cathepsin B. Immunostaining
for cathepsin B colocalizing with internalized beads indicates that
concanamycin did not affect the maturation of the phagosomes.
B, cells were pretreated with concanamycin as described
above. HRP-streptavidin (120 mg, 106 cells) was added to
cells for 30 min followed by a 1-h chase in MEM after washing four
times in MEM (no serum). Biotinylated collagen beads were added to
cells and incubated for varying times. Bead-associated proteins were
prepared as described under "Experimental Procedures" and
dot-blotted, and peroxidase activity due to streptavidin-biotin binding
was developed by ECL and quantitated by densitometry. Fusion was not
affected by pretreatment and the continuous presence of concanamycin
(n = 4). C, immunoblots of bead-associated
proteins in cells pretreated with concanamycin.
21 receptor-mediated
phagosomal maturation is partly dependent on elevated
[Ca2+]i.
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Fig. 10.
Role of calcium in bead internalization and
phagosomal acidification. A, cells were loaded with the
calcium indicator fura2/AM, and [Ca2+]i
measurements were done by ratio fluorimetry. Collagen-coated beads were
added at the time shown by the arrow in regular medium.
B, cells were incubated in Ca2+-free medium and
treated with 5 µM thapsigargin (time 0). Beads were added
at the time indicated by the arrow. Inhibition of calcium
uptake into intracellular stores blocks collagen bead-induced calcium
flux. C, in cells preloaded with 3 µM BAPTA/AM
(45 min), the addition of collagen beads (arrow) fails to
cause a calcium increase. D, cells preloaded with 3 µM BAPTA/AM for 45 min or treated with 5 µM
thapsigargin were incubated with FITC-streptavidin-biotin
collagen-coated beads. Calcium dependence of phagosomal acidification
was measured by ratio imaging as described in Fig. 7A.
Internalized beads were distinguished by the addition of
NH4Cl (40 mM). Data are mean ± S.E.
(n = 20).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
21
integrin-dependent collagen phagocytic system. Our study is
the first to characterize the protein composition of phagosomes and
some of the basic regulatory processes in the collagen degradative
pathway of fibroblasts.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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P. D. Arora, M.W.C. Chan, R. A. Anderson, P. A. Janmey, and C. A. McCulloch Separate Functions of Gelsolin Mediate Sequential Steps of Collagen Phagocytosis Mol. Biol. Cell, November 1, 2005; 16(11): 5175 - 5190. [Abstract] [Full Text] [PDF]
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V. M. Bhide, C. A. Laschinger, P. D. Arora, W. Lee, L. Hakkinen, H. Larjava, J. Sodek, and C. A. McCulloch Collagen Phagocytosis by Fibroblasts Is Regulated by Decorin J. Biol. Chem., June 17, 2005; 280(24): 23103 - 23113. [Abstract] [Full Text] [PDF]
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Q. Wang, G. P. Downey, M. T. Herrera-Abreu, A. Kapus, and C. A. McCulloch SHP-2 Modulates Interleukin-1-induced Ca2+ Flux and ERK Activation via Phosphorylation of Phospholipase C{gamma}1 J. Biol. Chem., March 4, 2005; 280(9): 8397 - 8406. [Abstract] [Full Text] [PDF]
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P. D. Arora, M. Glogauer, A. Kapus, D. J. Kwiatkowski, and C. A. McCulloch Gelsolin Mediates Collagen Phagocytosis through a Rac-dependent Step Mol. Biol. Cell, February 1, 2004; 15(2): 588 - 599. [Abstract] [Full Text] [PDF]
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D. Wienke, J. R. MacFadyen, and C. M. Isacke Identification and Characterization of the Endocytic Transmembrane Glycoprotein Endo180 as a Novel Collagen Receptor Mol. Biol. Cell, September 1, 2003; 14(9): 3592 - 3604. [Abstract] [Full Text] [PDF]
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 Z. Wang, T. M. Leisner, and L. V. Parise Platelet {alpha}2{beta}1 integrin activation: contribution of ligand internalization and the {alpha}2-cytoplasmic domain Blood, August 15, 2003; 102(4): 1307 - 1315. [Abstract] [Full Text] [PDF]
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P. D. Arora, L. Silvestri, B. Ganss, J. Sodek, and C. A. G. McCulloch Mechanism of Cyclosporin-induced Inhibition of Intracellular Collagen Degradation J. Biol. Chem., April 20, 2001; 276(17): 14100 - 14109. [Abstract] [Full Text] [PDF]
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Q. Wang, K. S. Ko, A. Kapus, C. A. G. McCulloch, and R. P. Ellen A Spirochete Surface Protein Uncouples Store-operated Calcium Channels in Fibroblasts. A NOVEL CYTOTOXIC MECHANISM J. Biol. Chem., June 15, 2001; 276(25): 23056 - 23064. [Abstract] [Full Text] [PDF]
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J. M. Kruger, T. Fukushima, V. Cherepanov, N. Borregaard, C. Loeve, C. Shek, K. Sharma, A. K. Tanswell, C.-W. Chow, and G. P. Downey Protein-tyrosine Phosphatase MEG2 Is Expressed by Human Neutrophils. LOCALIZATION TO THE PHAGOSOME AND ACTIVATION BY POLYPHOSPHOINOSITIDES J. Biol. Chem., January 18, 2002; 277(4): 2620 - 2628. [Abstract] [Full Text] [PDF]
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