©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Low Density Lipoprotein Receptor-related Protein/-Macroglobulin Receptor Mediates the Cellular Internalization and Degradation of Thrombospondin
A PROCESS FACILITATED BY CELL-SURFACE PROTEOGLYCANS (*)

Irina Mikhailenko , Maria Z. Kounnas (§) , Dudley K. Strickland (¶)

From the (1) Holland Laboratory, Department of Biochemistry, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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/-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 Kof 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.


INTRODUCTION

The low density lipoprotein receptor-related protein/-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) .

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 -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) .

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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) .

To derive quantitative data regarding the interaction between TSP and LRP, homologous-ligand competition assays were performed. Fig. 3 A demonstrates that 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 Kof 9 n M. In separate experiments, utilizing different preparations of TSP, the Kvaried 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 Kof 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 I-TSP in Cultured Fibroblasts

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-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 10cells) 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 10cells 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.

Fig. 7B shows that heparitinase treatment of cultured fibroblasts significantly reduces the LRP-mediated degradation of 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 (10IU/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.




DISCUSSION

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 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.

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 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.

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- (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.

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, -macroglobulin-proteinase complexes (13, 14) , and complexes of uPA with protease nexin-1 (61) .

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.


FOOTNOTES

*
This work was supported in part by Grants HL50787 and GM42581 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an Individual National Research Service Award HL08744 from the National Heart, Lung, and Blood Institute.

To whom correspondence should be addressed: American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0726; Fax: 301-738-0794.

The abbreviations used are: LRP, low density lipoprotein receptor-related protein/-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.

F. Battey, I. Mikhailenko, and D. Strickland, unpublished observation.


ACKNOWLEDGEMENTS

We thank Molly Migliorini for obtaining the sequencing data, and also Dr. David Mann for helpful suggestions.


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