From the
Thrombospondin (TSP) is a cell and matrix glycoprotein that
interacts with a variety of molecules. Newly synthesized thrombospondin
is either incorporated into the extracellular matrix, or binds to the
cell surface where it is rapidly internalized and degraded
(McKeown-Longo, P. J., Hanning, R., and Mosher, D. F. (1984) J.
Cell Biol. 98, 22-28). In the current investigation we
identify the low density lipoprotein receptor-related
protein/
The low density lipoprotein receptor-related
protein/
Thrombospondin (TSP) is a large
glycoprotein that is a member of a class of adhesive proteins which
contain the sequence Arg-Gly-Asp (RGD)
(26) . This sequence is
responsible for mediating cellular attachment by interacting with
integrins, a family of cell-surface receptors
(27, 28) .
TSP supports attachment of several cultured cells, including human
endothelial cells, and human smooth muscle cells via a RGD and calcium
dependent mechanism
(29) . TSP is also found in the
TSP is synthesized by endothelial cells
(32) , fibroblasts
(33) , and smooth muscle cells
(34) , and newly synthesized thrombospondin is either
incorporated into the extracellular matrix, or binds to the cell
surface where it is rapidly internalized and degraded
(35) .
Since this process can be blocked by excess unlabeled TSP, an
interaction with a cell-surface receptor was suspected
(35) . In
the present paper we have identified this receptor as LRP. Our results
demonstrate a high affinity interaction between LRP and TSP, and
confirm that LRP mediates the internalization and subsequent
degradation of TSP in cultured fibroblasts.
To derive quantitative data
regarding the interaction between TSP and LRP, homologous-ligand
competition assays were performed. Fig. 3 A demonstrates that
Fig. 7B shows that heparitinase
treatment of cultured fibroblasts significantly reduces the
LRP-mediated degradation of
TSP is a cell and matrix glycoprotein that interacts with a
variety of molecules. These include structural and matrix proteins such
as collagen
(45) and fibronectin
(46) , and polyanionic
molecules such as heparin
(47) . In addition, TSP is an
inhibitor of plasmin
(48) and neutrophil elastase
(49) activity. Unlike some matrix adhesive proteins, such as
fibronectin, TSP is rapidly catabolized by fibroblasts
(35) ,
Chinese hamster ovary cells
(43) , and endothelial cells
(50) in a process mediated by a specific cell-surface
receptor(s). In the current investigation we have identified LRP as a
receptor responsible for the internalization and degradation of TSP. A
specific and high affinity interaction between purified LRP and TSP was
demonstrated by affinity chromatography, ligand blotting experiments,
and homologous competition experiments. The interaction of LRP with TSP
has properties that are characteristic of LRP's interaction with
many other ligands. These include high affinity and specific binding to
the LRP heavy chain, a requirement of divalent cations for binding, and
antagonism of binding by RAP. Our experiments also document that LRP
mediates the cellular uptake of
An important feature of TSP catabolism is that
cell-surface proteoglycans facilitate this process. This was first
demonstrated when mutant Chinese hamster ovary cells, defective in
glycosaminoglycan synthesis, were found unable to internalize and
degrade TSP
(43) . The present studies confirm the importance of
cell-surface proteoglycans in the catabolism of TSP by demonstrating
that treatment of cells with heparitinase markedly reduces the extent
of LRP-mediated TSP uptake and degradation. In agreement with these
observations is the fact that TSP catabolism is also sensitive to
heparin. The participation of cell-surface proteoglycans in TSP
catabolism resembles their role in promoting the LRP-mediated
catabolism of lipoprotein lipase
(18) , and tissue factor
pathway inhibitor
(51) . While the exact role that cell-surface
proteoglycans play in the catabolism of these ligands is not clear,
they may function to concentrate the ligands on the cell surface and
facilitate their internalization and degradation by presenting them to
LRP. This is somewhat analogous to the role proteoglycans play in
presenting basic fibroblast growth factor to its receptor
(52) .
A similar transfer mechanism has been described for the LRP-mediated
internalization of uPA-plasminogen activator inhibitor-1 complexes
(53) , which initially bind to the urokinase receptor, and are
then transferred to LRP for internalization.
Results from the
present investigation suggest that LRP is the major receptor
responsible for mediating the internalization and degradation of
The ability of
LRP to directly bind and mediate the catabolism of TSP indicates that
this receptor may play an important role in regulating TSP levels in
plasma. In addition, since LRP is expressed in smooth muscle cells, LRP
likely also regulates TSP levels in the vessel wall. The significance
of the degradative pathway for TSP is not fully apparent at present.
However, TSP appears to have a diverse role in regulating cellular
proliferation, adhesion, and migration. The TSP gene is an inducible
immediate-early response gene to platelet-derived growth factor in
smooth muscle cells
(54) , and monoclonal antibodies against TSP
can inhibit smooth muscle cell growth
(55) . Furthermore, TSP
has been shown to be an inhibitor of angiogenesis
(56) . During
murine development, TSP antigen is localized primarily in regions of
cellular proliferation, migration, and intercellular adhesion
(57) , suggesting some role for TSP in these processes. Finally,
TSP has been suggested to directly activate transforming growth
factor-
TSP has also recently been identified as an
inhibitor of plasmin
(48) , neutrophil elastase
(49) ,
and cathepsin G
(59) . TSP may therefore act as a proteinase
inhibitor, and minimize the effect of proteinases on matrix
degradation. In this regard, it is interesting to highlight that LRP
binds and mediates the internalization of several proteinases and
proteinase-inhibitor complexes. These include uPA
(12) , tPA
(11) , complexes of tPA
(60) and uPA
(53) with
plasminogen activator inhibitor-1,
In summary, the present investigation has shown that
TSP binds to and is internalized by LRP in cultured cells. The
efficient catabolism of TSP requires the participation of cell-surface
proteoglycans, which may function to concentrate TSP on the cell
surface. The degradation of TSP is likely to be an important component
of the mechanisms that control TSP levels.
We thank Molly Migliorini for obtaining the sequencing
data, and also Dr. David Mann for helpful suggestions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-macroglobulin receptor (LRP) as a receptor
responsible for mediating the internalization of TSP leading to its
degradation. LRP is a large cell surface receptor consisting of a
515-kDa heavy chain and an 85-kDa light chain proteolytically derived
from a 600-kDa precursor. A specific and high affinity interaction
between purified LRP and TSP was demonstrated by homologous ligand
competition experiments, where a K
of
3-20 n
M was measured using different preparations of
TSP. The binding of TSP to purified LRP was completely inhibited by the
39-kDa receptor-associated protein, a known antagonist of ligand
binding by LRP. Cultured fibroblasts rapidly internalize and degrade
I-labeled TSP via a receptor-mediated process. This
process is inhibited by receptor-associated protein and by antibodies
against LRP, indicating that LRP is mediating the cellular
internalization of TSP. Our studies also confirm that the efficient
catabolism of TSP requires the participation of cell surface
proteoglycans, since digestion of cells with heparitinase markedly
reduces the extent of LRP-mediated TSP degradation. The ability of LRP
to directly bind and mediate the cellular internalization and
degradation of TSP indicates that this receptor may play an important
role in the catabolism of TSP in vivo.
-macroglobulin receptor (LRP)
(
)
is a large cell-surface receptor that is expressed in a
variety of tissues (for reviews, see Refs. 1-3). LRP contains a
515-kDa heavy chain to which ligands bind and a non-covalently
associated 85-kDa light chain which contains the transmembrane and
cytoplasmic domains
(4) . LRP is a member of the LDL receptor
family which also includes the LDL receptor
(5) , the VLDL
receptor
(6) , the vitellogenin receptor
(7) , and gp330
(8, 9, 10) . LRP is found in many cells types,
and mediates the cellular uptake and subsequent degradation of
proteinases, such as tissue-type plasminogen activator (tPA)
(11) and urinary-type plasminogen activator (uPA)
(12) ,
proteinase-inhibitor complexes, such as
-macroglobulin-proteinase complexes
(13, 14) , and apolipoprotein E-
(15, 16) and lipoprotein lipase-enriched
-VLDL and VLDL,
respectively
(17, 18, 19) . Furthermore, LRP is
also known to facilitate the internalization of Pseudomonas Exotoxin A
(20) . A 39-kDa protein, termed the
receptor-associated protein (RAP) binds to LRP
(21) , gp330
(22) , and the VLDL receptor
(23) with high affinity,
and to the LDL receptor with weaker affinity
(24) . Once bound,
RAP antagonizes ligand binding by members of this receptor family, and
may function to modulate ligand binding in vivo (21, 25) .
-granules of platelets
(30) , and is secreted when
platelets are activated with thrombin. After release from the
platelets, TSP binds to the platelet surface, where it may be involved
in mediating interactions with other platelets, or the endothelial
substratum
(31) .
Proteins
Thrombin was purchased from Enzyme
Research Laboratories, Inc. (South Bend, IN). Thrombospondin was
purified from thrombin-activated platelets essentially as described
(36) . Platelet concentrates were obtained from the American Red
Cross Blood Services and were washed and activated with thrombin as
described
(36) . After 2 min, thrombin activity was inhibited
with phenylmethylsulfonyl fluoride or with
D-Phe-Pro-Arg-CHCl (PPACK). The platelets were
pelleted, and the supernatant was concentrated, and applied to a
Sephacryl S-200 column (3
50 cm) equilibrated with 10 m
M Tris, 148 m
M NaCl, pH 7.4 (TBS). TSP from the gel
filtration column was applied to a heparin-Sepharose column (1.2
5 cm) equilibrated in TBS containing 5 m
M EDTA.
Following washing, TSP was eluted with 10 m
M Tris, pH 7.4,
containing 0.55
M NaCl. Peak fractions were pooled, dialyzed
against TBS, and stored at -70 °C. The concentration of TSP
was determined using an E
of 10.9
(37) . Thrombospondin was labeled with
[
I]Iodine using IODO-GEN (Pierce) following a
protocol previously described
(12) . The specific activity of
the iodinated protein was between 0.2 and 0.6 µCi/µg. LRP was
purified from human placenta as described
(13) , while RAP was
produced using a previously described expression system
(21) .
Bovine serum albumin and heparin were purchased from Sigma.
LRP-Sepharose Affinity
Chromatography
LRP-Sepharose was prepared by coupling human LRP
to CNBr-activated Sepharose (Pharmacia) at 1 mg/ml resin. Supernatant
from thrombin-stimulated platelets obtained from 5 units of platelets
(30 ml) was applied first to CL-4B Sepharose, then applied to an
LRP-Sepharose column (2 ml) and incubated for 1 h at room temperature.
The column was washed with 50 column volumes of 50 m
M Tris,
150 m
M NaCl, pH 8.0, then eluted with 2 column volumes of 8
M urea, 50 m
M Tris, pH 7.4. Eluted fractions were
subjected to SDS-PAGE in the absence of reducing agents on 4-12%
polyacrylamide gradient gels (Novex, San Diego, CA). Protein sequencing
analysis was performed on the affinity selected protein by first
eluting the protein bands from SDS-PAGE then sequencing the
amino-terminal using a Hewlett-Packard (model G1000S) protein
sequenator.
Antibodies
Mouse monoclonal anti-human TSP A6.1
(38) was utilized for ligand blotting studies. Antibodies to
human LRP (R777) were prepared as described
(20) and were
affinity-selected from rabbit polyclonal antisera using an
LRP-Sepharose column. Antibodies to the cytoplasmic domain of LRP were
prepared against a synthetic peptide and their preparation and
characterization have been previously described
(20) .
Binding Assays
Heterologous and homologous ligand
competition assays were performed as described by Williams et al. (21) . The computer program LIGAND
(39) was used to
analyze the data.
Ligand Blotting
Ligand blotting experiments were
carried out as described previously
(12) , with slight
modifications. Briefly, 3 µg of LRP was subjected to SDS-PAGE on
4-12% gradient gels (Novex) under nonreducing conditions, and
transferred to nitrocellulose filters. The filters were blocked with 3%
non-fat milk, and incubated with 50 n
M thrombospondin for 3 h
at room temperature in TBS containing 3% non-fat milk, 5 m
M CaCl, and 0.05% Tween 20. In some experiments, 400
n
M RAP was included. In those experiments examining the effect
of EDTA on the binding of TSP to immobilized LRP, the non-fat milk was
dialyzed overnight against TBS containing 10 m
M EDTA prior to
the assay. After three washes, the filters were incubated with a
anti-TSP monoclonal antibody (1.0 µg/ml) for 1 h at 25 °C, and
then incubated with a goat anti-mouse IgG-horseradish peroxidase
conjugate for 1 h at 25 °C. TSP binding was visualized using the
Renaissance Chemiluminescence kit (DuPont NEN, Boston, MA).
Cell Internalization and Degradation Assays
WI-38
human lung fibroblasts (ATCC CCL75, American Type Culture Collection,
Rockville, MD) were seeded into 12-well culture dishes 2-3 days
prior to assay and were grown in Dulbecco's modified
Eagle's medium (Mediatech, Washington, D.C.) supplemented with
10% bovine calf serum (Hyclone, Logan UT) and penicillin/streptomycin.
All experiments were performed using confluent cell layers. Surface
binding, internalization, and degradation of I-TSP
(generally 10 n
M, 50-150 cpm/fmol) by cells was measured
after incubation for indicated time intervals at 37 °C in 0.5 ml of
Dulbecco's modified medium containing 0.3 mg/ml bovine serum
albumin. Surface binding and internalization is defined as
radioactivity that is sensitive or resistant, respectively, to release
from cells by trypsin (0.5 mg/ml) and proteinase K (0.5 mg/ml) (Sigma)
in buffer containing 5 m
M EDTA. Degradation is defined as
radioactivity in the medium that is soluble in 10% trichloroacetic
acid. In all experiments a control was included in which the amount of
degradation products generated in the absence of cells was also
measured.
Heparitinase Treatment of Cells
Cells were
incubated with medium containing heparitinase (Seikagaku Corp., Tokyo,
Japan) at a concentration of 0.001 IU/ml for 45 min at 37 °C
followed by two rapid washes prior to internalization assays. In
control experiments, heparan sulfate (0.9 mg/ml) (Sigma) was pre-mixed
with the heparitinase to saturate the enzyme and protect cell-surface
proteoglycans from cleavage.
TSP Released from Thrombin-stimulated Platelets Binds
to LRP-Sepharose
Previous studies
(35) have suggested
that cultured fibroblasts contain a specific cell-surface receptor that
mediates the internalization of TSP, leading to its degradation. Since
LRP binds numerous ligands, and is expressed in fibroblasts, affinity
chromatography experiments were initiated to characterize the potential
interaction between TSP and LRP. TSP is a component of the platelet
-granule, and is released when platelets are activated with
thrombin. In the present experiments, the supernatant from
thrombin-stimulated platelets was applied to LRP-Sepharose. After
extensive washing, the column was eluted in a buffer containing 8
M urea. Selected fractions were analyzed by SDS-PAGE under
nonreducing conditions (Fig. 1). A major polypeptide, with an
approximate molecular mass of 400 kDa, was eluted from the affinity
resin. Upon reduction, a polypeptide with an apparent molecular mass of
160 kDa was noted (data not shown). Amino-terminal sequencing of the
band excised from the gel yielded the sequence of
Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn, which corresponds to the
amino-terminal sequence of human TSP
(26) . The polypeptide
eluted from the LRP-Sepharose column also reacted with a monoclonal
antibody against TSP upon immunoblotting following transfer to
nitrocellulose (data not shown). These data confirm that TSP binds to
immobilized LRP-Sepharose.
TSP Binds to the LRP Heavy Chain and Its Binding Is
Prevented by RAP
To further characterize the interaction between
TSP and LRP, ligand blotting experiments were employed. The results
(Fig. 2) demonstrate that TSP, like other LRP ligands, binds to the
heavy chain of LRP. The amount of TSP bound was significantly reduced
by including RAP during the incubation. RAP is known to prevent the
binding of ligands to LRP
(21, 25) . Furthermore, the
binding was reduced when EDTA was added to the incubation mixture,
suggesting a requirement for divalent cations for the interaction.
While TSP itself binds calcium
(40) , the requirement of metal
ions for the binding of this protein to immobilized LRP is in agreement
with the known requirement of metal ions for the binding of most
ligands to LRP
(13, 41) .
I-TSP binds to microtiter wells coated with purified LRP,
but not microtiter wells coated with bovine serum albumin. The binding
is prevented by excess cold TSP, which is consistent with specific
binding. The data are adequately described by a model containing a
single class of sites with a K
of 9
n
M. In separate experiments, utilizing different preparations
of TSP, the K
varied between 3 and 20
n
M. Fig. 3 B demonstrates that RAP inhibits the
binding of
I-TSP to microtiter wells coated with LRP with
a K
of 0.5 n
M, a value that is
in excellent agreement with the known affinity of LRP for RAP
(21) . Together, these results indicate a high affinity and
specific binding of
I-TSP to LRP, and document that RAP
prevents this interaction.
LRP Mediates the Internalization and Degradation of
Since our
experiments have documented a high affinity and specific interaction
between TSP and LRP, it was of interest to determine whether or not LRP
is capable of mediating the cellular internalization of
I-TSP in Cultured Fibroblasts
I-labeled TSP. The role of LRP in TSP uptake and
degradation was investigated using RAP and affinity purified antibodies
against LRP which are known to block ligand internalization
(12, 18, 42) . Fig. 4 demonstrates the time
course of surface binding, internalization, and degradation of
I-TSP by cultured fibroblasts at 37 °C, and the
effect of RAP on this process. When added to cultured fibroblasts at 37
°C,
I-TSP demonstrated a slow, time-dependent binding
to cells that was not blocked by RAP ( top panel,
Fig. 4
). This binding was also not competed by addition of excess
unlabeled TSP (data not shown). In contrast, RAP inhibits the
internalization ( middle panel, Fig. 4) and the
degradation ( bottom panel, Fig. 4) of
I-TSP.
Similar results were obtained in two additional experiments, each
utilizing different TSP preparations. The effect of RAP concentration
on the internalization and degradation of
I-labeled TSP
is shown in Fig. 5. The data reveal that relatively low concentrations
of RAP are effective in inhibiting the internalization and degradation
of
I-labeled TSP, and that RAP is more effective than
unlabeled TSP in blocking internalization and degradation of
I-labeled TSP. These results confirm that a RAP-sensitive
receptor, most likely LRP, mediates the cellular uptake and degradation
of
I-TSP in cultured fibroblasts.
Figure 4:
Effect of RAP on the time course of
surface binding, internalization, and degradation of
I-TSP by WI-38 cells. Wells containing WI-38 fibroblasts
(1
10
cells) were incubated at selected time
intervals with
I-TSP (10 n
M) in the absence or
presence of unlabeled RAP (400 n
M). At selected time intervals
the medium was removed, and trichloroacetic acid added. The
radioactivity present in the supernatant was measured to determine the
amount of degraded
I-TSP. To determine the surface
binding and internalization, the cells were washed with cold buffer,
then incubated with trypsin-EDTA + 0.5 mg/ml proteinase K to
detach the cells from the well and to dissociate surface-bound ligand.
The cells were collected by centrifugation and radioactivity associated
with cells was defined as internalized TSP, while surface-bound ligand
was defined as the radioactivity released from cells by
trypsin-EDTA-proteinase K treatment.
,
I-TSP;
,
I-TSP + RAP. Each data point represents
the mean of triplicate determinations.
Since RAP is known
to interact with the VLDL receptor and gp330, confirmation for the role
of LRP in the internalization and degradation of TSP was obtained by
the use of specific LRP antibodies which are known to block the uptake
and degradation of ligands. Fig. 6 demonstrates that an affinity
purified anti-LRP IgG is effective in preventing the internalization
and degradation of I-TSP by cultured fibroblasts. As a
control, an antibody against the cytoplasmic domain of LRP was
utilized, and was unable to inhibit either the internalization or the
degradation of
I-TSP (Fig. 6). These experiments
confirm that LRP is responsible for mediating the internalization of
I-TSP leading to its degradation.
Figure 6:
Anti-LRP IgG inhibits I-TSP
internalization ( A) and degradation ( B). Wells
containing 1
10
cells were incubated with
I-TSP (10 n
M) for 5 h at 37 °C in the
absence or presence of RAP (400 n
M), anti-LRP IgG (0.1 mg/ml),
or anti-LRP cytoplasmic domain ( CD) antibodies (0.1 mg/ml).
The internalization ( A) and degradation ( B) of
I-TSP were determined as described in legend to Fig. 4.
Each data point represents the mean of triplicate
determinations.
Proteoglycans Facilitate the Internalization and
Degradation of TSP
Our data demonstrate that LRP is responsible
for mediating the cellular internalization of TSP by WI-38 fibroblasts.
However, it is also apparent that TSP binds to molecules other than LRP
on the cell surface. Previous studies have reported a reduction in the
binding and degradation of TSP by Chinese hamster ovary cells defective
in glycosaminoglycan synthesis
(43) . In order to determine if
cell-surface proteoglycans facilitate TSP catabolism in cultured
fibroblasts, cells were first treated with heparitinase, and then the
ability of control and treated cells to bind I-labeled
TSP at 4 °C measured. The results of this experiment are shown in
Fig. 7 A, and demonstrate that both heparin and heparitinase
treatment of fibroblasts reduces the amount of
I-TSP
bound to the cells at 4 °C. In a control experiment, heparan
sulfate was added separately and along with heparitinase. Heparan
sulfate, a substrate for the enzyme, by itself had no effect on
I-TSP binding to cells. However, exogenous heparan
sulfate did minimize the effect of heparitinase treatment on
I-TSP binding. This control experiment suggests that the
decrease in
I-TSP binding after heparitinase treatment of
cells is very likely the result of removal of cell-surface
proteoglycans.
I-TSP at 37 °C.
Heparitinase treatment of cultured fibroblasts had a similar effect on
the uptake of TSP by fibroblasts (data not shown). These results
confirm that cell-surface proteoglycans play an important role in TSP
catabolism and appear to be required for the LRP-mediated
internalization and degradation of TSP in cultured fibroblasts.
Figure 7:
Effect of heparin and heparitinase
treatment of cells on I-TSP binding at 4 °C (A), or
I-TSP degradation at 37 °C (B). WI-38 fibroblasts (1
10
) were incubated with or without heparitinase
(10
IU/ml) for 45 min at 37 °C. The cells were
then washed and used for the assays. A, cells were chilled to
4 °C, and incubated with
I-TSP (10 n
M) in
the absence or presence of RAP (400 n
M) or heparin (20
µg/ml) for 2 h. After incubation, the cells were washed, and the
amount of radioactivity associated with cells determined. In indicated
experiments, heparan sulfate ( HS) (0.9 mg/ml) was pre-mixed
with heparitinase prior to adding the enzyme to the cells. B,
control cells or cells treated with heparinase were incubated with
I-TSP (2 n
M) in the absence or presence of RAP
(400 n
M), unlabeled TSP (600 n
M), or heparin (20
µg/ml) for 7 h at 37 °C. After this period, the extent of
degradation of
I-TSP was determined as described in the
legend to Fig. 4. Each data point represents the mean of triplicate
determinations.
Fate of Surface Bound TSP
The previous experiments
document the importance of cell-surface proteoglycans in the
LRP-mediated degradation of TSP. In addition to its interaction with
proteoglycans, TSP is known to bind to a number of different
cell-surface molecules (for review, see Ref. 44). It was of interest
therefore to determine the fate of I-TSP bound to the
cell surface, and to investigate the fraction of cell-surface bound
protein that is actually internalized and degraded. To measure this,
I-TSP was incubated with cultured fibroblasts at 4 °C
in the absence and presence of RAP. After removal of unbound ligand by
washing, the temperature was raised to 37 °C to initiate
endocytosis, and the cells incubated in the presence or absence of RAP.
At selected time intervals, the fate of the surface bound
I-TSP was followed. The results of these experiments are
shown in Fig. 8. In the absence of RAP, approximately 80% of the
I-TSP disappeared from the surface of the cell with time
( top panel, Fig. 8). A portion of the
I-TSP simply dissociates from the cell surface and is
released into the culture medium (data not shown). However,
approximately 35-40% of the labeled material is internalized
( middle panel, Fig. 8) and eventually degraded
( bottom panel, Fig. 8). In the presence of RAP,
significantly less material was internalized (Fig. 8, middle
panel), confirming the role of LRP in this process. RAP also
reduced the amount of
I-TSP that was degraded ( bottom
panel, Fig. 8). The results of these experiments confirm
that a substantial portion of the
I-TSP bound on the cell
surface is rapidly internalized and degraded at 37 °C in a process
mediated by LRP.
Figure 8:
Distribution of I-TSP with
time during endocytosis in cultured human lung fibroblasts in the
absence and presence of RAP. WI-38 fibroblasts were incubated with 10
n
M
I-TSP for 2 h at 4 °C to allow surface
binding in the absence and presence of RAP (400 n
M). After
washing, media was changed and cells were incubated at 37 °C in the
absence or presence of RAP (400 n
M). At selected time
intervals the surface binding, internalization, and degradation of
I-TSP were determined as described in the legend for Fig.
4.
,
I-TSP;
,
I-TSP +
RAP. Each data point represents the mean of triplicate
determinations.
I-labeled TSP in cultured
fibroblasts leading to its degradation. This has been confirmed by
demonstrating that RAP and anti-LRP antibodies prevented the
degradation of
I-labeled TSP by these cells. It should be
pointed out that RAP is an effective inhibitor of ligand binding to
LRP, gp330, and the VLDL receptor, and thus RAP is not a specific
antagonist for LRP. Our data indicate that in lung fibroblasts, LRP is
the major receptor responsible for TSP catabolism, since anti-LRP
antibodies are as effective as RAP in blocking degradation of
I-TSP.
I-TSP in fibroblasts. This is based on the observation
that anti-LRP antibodies are able to completely inhibit the degradation
of
I-TSP in these cells. Furthermore, it appears that
cell lines that lack LRP are greatly reduced in their capacity to
internalize and degrade TSP. For example, our own studies
(
)
found that cultured human umbilical vein endothelial cells
are unable to internalize or degrade significant amounts of
I-labeled TSP. When detergent extracts of human umbilical
vein or human aorta endothelial cells were analyzed by immunoblotting,
no LRP antigen was detected. Cellular uptake experiments revealed that
I-labeled
-macroglobulin-proteinase
complexes are not internalized by these cells. These studies confirm
that human umbilical vein endothelial cells and human aortic
endothelial cells lack detectable LRP. Very likely, this accounts for
their greatly reduced capacity to internalize and degrade TSP. While
our studies suggest that LRP is the major receptor responsible for the
cellular internalization of TSP, it is certainly possible that other
cell-surface molecules may also mediate the internalization and
degradation of TSP. In contrast to our studies with human umbilical
endothelial cells, Murphy-Ullrich and Mosher
(50) reported that
normal and variant bovine aorta endothelial cells were capable of
internalizing and degrading
I-labeled TSP. What is not
known at present is whether or not these cell lines express LRP. If so,
then this would explain their ability to internalize and degrade TSP.
Alternatively, if they do not contain LRP, then it is likely that
another receptor, perhaps another LDL receptor family member, is also
able to mediate the cellular internalization of TSP.
(58) , a potent growth regulatory protein normally
secreted from cells in a latent form. All of these studies suggest that
TSP may impact a variety of biological processes, and if so, it would
be important to have a mechanism for rapid catabolism of TSP to
regulate its level.
-macroglobulin-proteinase complexes
(13, 14) , and complexes of uPA with protease nexin-1
(61) .
-macroglobulin receptor; LDL, low density
lipoprotein; VLDL, very low density lipoprotein, gp330, glycoprotein
330; RAP, receptor-associated protein; TSP, thrombospondin; PAGE,
polyacrylamide gel electrophoresis; uPA, urinary-type plasminogen
activator; tPA, tissue-type plasminogen activator.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.