Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M602919200 on August 25, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31562-31571, October 20, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/42/31562    most recent
M602919200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brandan, E.
Right arrow Articles by Marzolo, M.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brandan, E.
Right arrow Articles by Marzolo, M.-P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Low Density Lipoprotein Receptor-related Protein Functions as an Endocytic Receptor for Decorin*

Enrique Brandan1, Claudio Retamal, Claudio Cabello-Verrugio2, and María-Paz Marzolo3

From the Centro de Regulación Celular y Patología "Joaquín V. Luco," CRCP, Departamento de Biología Celular y Molecular, MIFAB, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

Received for publication, March 28, 2006 , and in revised form, July 5, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Decorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type beta and other growth factors and thereby influences the processes of proliferation and differentiation in a wide array of physiological and pathological reactions. Hence, understanding the regulatory mechanisms of decorin activity has broad implications. Here we report that the extracellular levels of decorin are controlled by receptor-mediated catabolism, involving the low density lipoprotein receptor family member, low density lipoprotein receptor-related protein (LRP). We show that decorin is endocytosed and degraded by C2C12 myoblast cells and that both processes are blocked by suppressing LRP expression using short interfering RNA. The same occurs with CHO cells, but not with CHO cells genetically deficient in LRP. Finally, we show that LRP-null CHO cells, transfected to express mini-LRP polypeptides containing either the second or fourth LRP ligand-binding domains, carry out decorin endocytosis and lysosomal degradation. These findings point to LRP-mediated catabolism as a new control pathway for the biological activities of decorin, specifically for its ability to influence extracellular matrix signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Decorin is one of the most studied members of the family of small leucine-rich proteoglycans. Its core protein, which constitutes up to 80% of the protein moiety, is composed of 12-fold repeats of a 24-amino acid residue (leucine-rich repeats). In addition, decorin carries a single glycosaminoglycan (GAG)4 chain at its NH2 terminus. A crystal structure for bovine decorin has been published (1) that together with earlier x-ray scattering data (2) suggests decorin to be a dimeric protein. Each monomer adopts a curved structure, whereby antiparallel dimerization occurs through the beta-sheet on the monomer's concave surface.

Several different functions, based on the interaction of the core protein with other proteins, have been established for decorin, one example being the regulation of extracellular matrix (ECM) assembly. Decorin regulates collagen fibril formation and stabilization and also modulates cell adhesion (3). The interaction of decorin with fibronectin and thrombospondin leads to the inhibition of fibroblast attachment to these substrata (4, 5). In addition to the interaction with ECM constituents, decorin interacts with several growth factors and plasma membrane-located receptors. For instance, it is well known that decorin has the ability to form complexes with transforming growth factor type-beta (TGF-beta) (6), bind to the insulin-like growth factor-I (7), and interact with tumor necrosis factor-{alpha} (8). Furthermore, the ectopic expression of decorin has been shown to retard the growth of various tumor cells, an effect that can be attained by exogenously applying recombinant decorin to a wide variety of cells (9-12). Decorin is also known to cause rapid phosphorylation of the epidermal growth factor receptor and concurrent activation of the mitogen-activated protein kinase signaling pathway (13). Recent studies have shown that decorin binds to the insulin-like growth factor-I receptor, inducing its phosphorylation and activation, followed by receptor down-regulation (7). On the other hand, reducing decorin levels results in a decreased cell responsiveness to TGF-beta, suggesting that decorin is required to activate the TGF-beta signaling pathways (14).

Considering the different ECM-related functions exhibited by decorin, including the accumulation of growth factors and its interaction with matrix constituents as well as several transducing receptors at the cell surface, it is evident that the regulation of extracellular decorin concentrations, by varying its biosynthesis and degradation rates, is of great physiological importance.

The metabolism of decorin has been studied most intensively in cultured fibroblasts, in which decorin represents the major proteoglycan species and is secreted into the culture medium, where it follows secretion-recapture cycles (15). Fibroblasts and other cells of mesenchymal origin are known to efficiently internalize decorin by receptor-mediated endocytosis (16, 17). Several concerted yet unsuccessful efforts have been made to identify the endocytic receptor of decorin. Two proteins of 51 and 26 kDa, present in endosomes and at the plasma membrane, are considered putative decorin receptors (18). However, no functional evidence is as yet available to support this notion, so that the identity of the decorin receptor remains an open question.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 1.
Decorin is endocytosed by C2C12 myoblasts. A, C2C12 myoblasts were incubated with [35S]decorin at 37 °C for 3 h in the absence (Ctrl) or presence of either decorin (Dcn) or heparin (Hep). After this, cells were analyzed to determine the extent of [35S]decorin endocytosis (black bars) and degradation (gray bars). Endocytosis is expressed as the clearance of [35S]decorin (16): volume/mg of protein/h. Degradation values, expressed as percentages, were normalized against control cells. B, C2C12 myoblasts were incubated with [125I]decorin core protein (Dcn core) for 3 h at 37 °C, in the absence (Control) or presence of unlabeled decorin core protein (Dcn), heparin (Hep), biglycan (Big), or chloroquine (Chloroq). Degraded decorin core protein is expressed as fmol of [125I]decorin/105 cells.

 
The low density lipoprotein (LDL) receptor-related protein, LRP, is a giant receptor belonging to the LDL receptor family, which binds, endocytoses, and mediates the degradation of several ligands (19). The receptor's folding process in the endoplasmic reticulum requires the participation of the 39-kDa receptor chaperone, RAP (20). This chaperone protein has also been used as a tool and competitor to study novel ligands for LRP, since recombinant RAP binds with high affinity to the receptor's ligand-binding domains at the cell surface.

Through its large ectodomain, which contains four ligand-binding domains, LRP binds (among other proteins) multiple ECM molecules, including thrombospondin (21-23), fibronectin (24), plasminogen activators (25, 26), matrix metalloproteinases (27), and connective tissue growth factor (28). Furthermore, LRP regulates signaling cascades by binding ECM molecules, such as fibronectin (29) and thrombospondin (30), and growth factors, such as platelet-derived growth factor (31, 32), connective tissue growth factor (33), and TGF-beta (34, 35). Interestingly, several of these molecules also bind decorin (36-39).

Given the role of decorin in myoblast differentiation (14, 40) and the presence of LRP mRNA in human skeletal muscle cells (41), in the present study, we tested whether decorin endocytosis in C2C12 mouse myoblasts was affected by the presence of the RAP-inhibitable receptor, LRP. Our results showed unequivocally that in LRP-expressing C2C12 myoblasts, the internalization and degradation of decorin depended on its interaction with this endocytic receptor. Furthermore, we also demonstrated that in Chinese ovary cells (CHO), LRP was also responsible for decorin endocytosis, involving at least the receptor's ligand-binding domains 2 and 4.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Plasmids encoding LRP minireceptors, which include the ligand-binding domains 2 (mLRP2) and 4 (mLRP4) have been described previously (42, 43). Annealed LRP-1-specific siRNA as well as control siRNA were obtained from Ambion (Austin, TX), with LRP siRNA sequences described in Ref. 44. GST and GST-RAP were produced as described in (45). Rabbit anti-LRP antibody was kindly provided by Dr. Guojun Bu and used as prescribed by Marzolo et al. (42). Mouse anti-LRP raised against the cytoplasmic domain was purchased from Calbiochem, and mouse anti-{alpha}-tubulin was from Sigma. Adenoviral vector containing the full-length cDNA for human decorin (Adv-Dcn) has already been described (14). Decorin core protein was obtained from R&D Systems, and full-length decorin and biglycan were purchased from Sigma. To determine the amount of chondroitin and dermatan sulfate in the commercial decorin, decorin was radiolabeled with 125Ias explained below and treated with chondroitinase ABC and AC. Both treatments digested most if not all of the GAGs associated to decorin, indicating its chondroitin sulfate nature. The cross-linker agent disuccinimidylsuberate (DSS) was from Pierce.

Cell Culture and Transfection—The mouse skeletal muscle cell line C2C12 (ATCC) (46) was grown and induced to differentiate, as described in Ref. 47. The U87 glioblastoma cell line was cultured in minimal essential medium with 0.1% nonessential amino acids and 1 mM sodium pyruvate. Wild-type Chinese hamster ovary cells (CHO-K1) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), whereas LRP-deficient cells (LRP-null CHO) (48) were cultured in F-12 medium with 10% FBS. Clonal cell lines, derived from LRP-null CHO cells expressing LRP minireceptors, were obtained by transfection, using 2 µg of plasmid DNA and Lipofectamine Plus transfection reagent (Invitrogen) in 35-mm dishes, according to the supplier's protocol. Cells were screened and analyzed by Western blot and immunofluorescence. Selected clones were then maintained in wild-type medium containing 0.4 mg/ml G418.

RNA Isolation and Reverse Transcription (RT)-PCR—Total RNA was isolated from myoblast cultures using Trizol (Invitrogen). For RT reactions, 4 µg of total RNA were treated with DNase I for 15 min at room temperature. Subsequently, samples were incubated with random hexamers and the Maloney murine leukemia virus reverse transcriptase kit for 10 min at 25 °C, 60 min at 37 °C, and finally 10 min at 70 °C. Aliquots of 1 µl of cDNA were used as a template for standard PCR procedures. The primers used in PCR reactions were as follows: LRP forward, 5'-AGTGCTGCCCAGACACAGCTCAAGTGTG-3'; LRP reverse, 5'-CACGATCTTGCTATCCACCAGCTTGGTG-3'; glyceraldehyde-3-phosphate dehydrogenase forward, 5'-CGGTGTGAACGGATTTGGC-3'; glyceraldehyde-3-phosphate dehydrogenase reverse, 5'-GCAGTGATGGCATGGACTGT-3'.

C2C12 Infection with Recombinant Adenovirus, Adv-Dcn, and Metabolic Labeling—C2C12 myoblasts were plated at a density of 30,000 cells/cm2 in 6-well plates. After 4 h, myoblasts were infected with 500 plaque-forming units/cell of Adv-Dcn (14) in DMEM, containing 2% heat-inactivated FBS. After 90 min of incubation, standard medium was added, and incubation continued for an additional 24 h, after which cells were metabolically labeled for 18 h in sulfate and serum-free DMEM/F-12, containing 100 µCi/ml [35S]H2SO4 (25 mCi/ml; PerkinElmer Life Sciences). This conditioned medium was then removed, concentrated, and partially purified on a DEAE-Sephacel column, pre-equilibrated in 10 mM Tris-HCl, pH 7.4, 0.2 M NaCl, and 0.1% Triton X-100. Column-bound samples were incubated with heparitinase, in appropriate buffer for 4 h at 37 °C, in order to degrade any heparan sulfate proteoglycans present in the conditioned medium. The DEAE-Sephacel was incubated with 1 M NaCl, and the eluate was dialyzed against phosphate-buffered saline. In experiments using decorin core protein, the carrier-free decorin core protein (R&D Systems) was radiolabeled with Na[125I] using chloramine T (49).


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 2.
Decorin endocytosis is inhibited by RAP in skeletal muscle cells. A, myoblasts were incubated with [35S]decorin at 37 °C for 3 h, in the absence (Control) or presence of either GST or GST-RAP. After this, cells were analyzed to determine [35S]decorin endocytosis (black bars) and degradation (gray bars), as for Fig. 1. Endocytosis is expressed as the clearance of [35S]decorin (i.e. volume/mg of protein/h). Degradation values, expressed as percentages, were normalized against control cells. B, myoblasts were incubated with [125I]decorin core protein (Dcn core) at 37 °C for different time periods in the presence of GST-RAP (white circles) or alone (black circles). Internalized and degraded decorin levels were determined as for Fig. 1.

 
siRNA Transfection—For transfection experiments, cells were plated in 6-well plates and incubated until reaching 70% confluence. Cells were subsequently incubated for 6 h in 800 µl of Opti-MEM I, containing 75 nM LRP siRNA or control siRNA plus 8 µl of Lipofectamine 2000 (Invitrogen). Following this transfection period, FBS was added to the medium, and the cells cultured for a further 12 h. The medium was then changed to growth medium, and cells were incubated up to 72 h post-transfection, after which decorin endocytosis, degradation, or cross-linking assays were carried out. The effect of siRNA on the level of LRP synthesis in C2C12 myoblasts was evaluated by immunoblotting against LRP (50).

Cross-linking and Competition Assays—Briefly, cells were incubated with 220 pM [125I]decorin for 4 h at 4 °C in KRH buffer (128 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.3 mM CaCl2, 50 mM Hepes, pH 7.4) supplemented with 0.5% bovine serum albumin (KRH-bovine serum albumin). In competition experiments, cells were co-incubated with [125I]decorin and either recombinant decorin core protein (22 nM), bovine decorin containing GAGs (22 nM), heparin (100 µg/ml), GST (1 µM), or GST-RAP (1 µM). The cells were then sequentially washed in cold KRH-bovine serum albumin and KRH. For cross-linking assays, cells were incubated with cross-linker agent DSS in KRH buffer for 30 min at 4 °C. The reaction was stopped by adding a buffer of 10 mM Tris-HCl, pH 7.4, containing 250 mM sucrose (49). In immunoprecipitation experiments, DSS was omitted. Cells were lysed in 50 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 0.5% Triton X-100, containing a mixture of protease inhibitors and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (80 µg) from precleared extracts were separated by SDS-PAGE in 3-8% gradient gels, and gels were finally dried and exposed under a PhosphorImager.

Confocal Immunofluorescence Microscopy—LRP expression and distribution in C2C12 was analyzed by confocal microscopy. Cells were grown on glass coverslips. For intracellular protein staining, cells were fixed in 3% paraformaldehyde, permeabilized with 0.05% Triton X-100 (51), and incubated for 1 h with 1:100 mouse anti-LRP antibody, directed against the cytoplasmic tail of human LRP-1 (Calbiochem). The incubation buffer was 50 mM Tris-HCl, pH 7.7, 0.1 M NaCl, and 2% bovine serum albumin. After buffer removal and several washes with the above buffer, bound antibodies were detected by incubating the cells for 30 min with 1:100 affinity-purified fluorescein iso-thiocyanate-conjugated anti-mouse antibodies (Pierce). After rinsing, the slides were viewed under a Pascal Zeiss laser-scanning confocal microscope (LSM-5).

Immunoprecipitation and Immunoblot Analyses—For immunoprecipitation assays, myoblasts were lysed in 50 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 0.5% Triton X-100 buffer, containing a mixture of protease inhibitors and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (150 µg) from precleared extracts were immunoprecipitated overnight at 4 °C with 5 µg of rabbit anti-LRP, as previously described (50), followed by incubation for 2 h at 4 °C with 20 µl of protein A-agarose beads (Pierce). Equal volumes of immunoprecipitated protein were subjected to SDS-PAGE in 3-8% gradient gels, which were then dried and exposed to phosphorimaging or subjected to LRP immunoblot analysis.


Figure 3
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3.
Core protein and glycosaminoglycan chain of decorin both participate in its endocytosis and degradation through LRP. Myoblasts were incubated with [35S]decorin at 37 °C for 3 h, in the absence (Ctrl) or presence of cold decorin (Dcn full), decorin core protein (Dcn core), chondroitin sulfate (CS), GST-RAP (RAP), decorin core plus chondroitin sulfate (Dcn core + CS), or decorin plus GST-RAP (Dcn full + RAP). After this, cells were analyzed to determine [35S]decorin endocytosis (A) and degradation (B). Endocytosis and degradation of decorin are expressed as explained in the legend of Fig. 1.

 
For immunoblot assays, cell extracts were prepared in the same Tris buffer as above, containing protease inhibitors and phenylmethylsulfonyl fluoride. Aliquots were separated by SDS-gel electrophoresis in 3-8% gradient polyacrylamide gels, electrophoretically transferred onto nitrocellulose membranes (Schleicher & Schuell) and probed with either rabbit anti-LRP (1:1000) or mouse anti-{alpha}-tubulin (1:5000) antibodies (Sigma). All immunoreactions were visualized by enhanced chemiluminescence (Pierce).

Decorin Endocytosis and Degradation Assays—The rates of [35S]decorin endocytosis and degradation were determined in myoblast cultures, in the absence of serum, as described (16). Briefly, cells were grown in 6-well plates until reaching 80% confluence, after which [35S]decorin (200,000 cpm) was added (in a total volume of 1 ml) and left for 3 h at 37 °C in the presence or absence of bovine decorin containing GAGs (100 nM), decorin core (100 nM), biglycan (100 nM), heparin (100 µg/ml), chondroitin sulfate (100 nM), GST (1 µM), or GST-RAP (1 µM). In some experiments, cells were treated with LRP siRNA, prior to adding labeled decorin. Since proteoglycan endocytosis is followed by intralysosomal degradation and the concomitant release of inorganic sulfate into the culture medium, endocytosis of [35S]decorin can be measured by determining the amounts of [35S]sulfate present both in the intracellular and culture medium. Soluble [35S]sulfate corresponding to inorganic sulfate was determined after precipitating [35S]decorin with 70% ethanol. Endocytosis can hence be expressed as a clearance rate: the volume of decorin-cleared medium as a function of time and cellular protein level. Degradation was defined as the sum of the intra- and extracellular levels of ethanol-soluble radioactivity over the total amount of endocytosed decorin.

For [125I]decorin core protein degradation assays, cells were seeded 18 h before the assay, at a density of 30,000 cells/cm2 in 12-well plates. They were then washed with phosphate-buffered saline, depleted for 1.5 h in serum-free medium, and washed with binding medium (DMEM/F-12 medium with 0.5% bovine serum albumin and 5 mM CaCl2). Incubations were carried out at 37 °C using 400 µl of binding medium containing 1 nM [125I]decorin and either competitors or controls, as indicated. Decorin degradation was calculated as the amount of non-trichloroacetic acid-precipitable radioactivity recovered in the medium after incubation, and cpm readings were then transformed to fmol using iodinated decorin-specific activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Decorin and Decorin Core Protein Are Internalized and Degraded by C2C12 Myoblasts—It has been proposed that decorin is endocytosed by several cell types, although the receptor responsible for this process has yet to be identified. In order to investigate this issue, we analyzed the endocytosis of decorin in C2C12 myoblasts. Fig. 1A shows that [35S]decorin, isolated and purified from C2C12 cells transfected with recombinant adenovirus containing the human decorin sequence, is indeed endocytosed. When expressed as the volume of cleared radio-active ligand per unit of time and protein concentration, myoblasts cleared about 50 µl/mg/h of [35S]decorin. These values are almost twice those reported for decorin clearance in fibroblasts by Hausser et al. (16). Fig. 1A (left) also shows that upon the addition of cold commercial decorin, isolated from cartilage, [35S]decorin clearance was inhibited by about 60%. Degradation of [35S]decorin by myoblasts was close to 80% of the total amount of decorin that was internalized (Fig. 1A, right). Again, these values are higher than those described for fibroblasts (16). Moreover, it has been previously demonstrated that [35S]decorin endocytosis and degradation decrease in the presence of heparin (52). In order to determine whether these processes were dependent on the GAG chains present in decorin, we repeated the experiments using a decorin core protein devoid of GAGs. Fig. 1B shows that this 125I-labeled decorin core protein was efficiently degraded in a process that was inhibited by unlabeled decorin core protein, commercial biglycan isolated from cartilage, heparin, and also the lysosomal inhibitor, chloroquine. These results indicate that C2C12 myoblasts were able to internalize and degrade whole decorin molecules as well as decorin core protein.


Figure 4
View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 4.
C2C12 myoblasts express LRP, the levels of which decrease during myogenesis. A, total RNA was obtained from C2C12 myoblasts and either treated (+RT) or not treated (-RT) with reverse transcriptase. RT-PCR analyses were performed using specific primers aimed at detecting either LRP or glyceraldehyde-3-phosphate dehydrogenase mRNA. Total RNA from U87 cells was used as a positive control. B, Western blot analyses were performed to determine the protein levels of LRP in C2C12 myoblast extracts. U87 cell extracts were used as positive controls, and tubulin protein levels are shown as loading controls. C, cells were permeabilized and processed for confocal indirect immunofluorescent staining, using anti-LRP antibodies and fluorescein isothiocyanate-conjugated secondary antibodies (a) and controls without anti-LRP antibodies (b). The bar corresponds to 10 µm. D, Western blots for LRP were performed on the cell extracts of myoblasts induced to differentiate for 0, 2, 4, or 6 days. U87 cell extracts were used as positive controls, and for all cells, tubulin protein levels provided a loading control.

 
Decorin Clearance and Degradation Are Inhibited by RAP—To gain more insight into the possible endocytic receptor(s) involved in the uptake of decorin, C2C12 myoblasts were incubated with [35S]decorin in the presence of GST-RAP, which prevents the association of several ligands to members of the LDL receptor family. Upon co-incubation with RAP, both [35S]decorin clearance and degradation were strongly inhibited, whereas incubating with GST alone had no effect (Fig. 2A). Fig. 2B shows the kinetics of [125I]decorin core protein internalization and degradation. A plateau for internalization was attained after 200 min of incubation, whereas degradation of [125I]decorin core protein continued up to 300 min. Both processes were inhibited by GST-RAP, suggesting that a member of the LDL receptor family could be involved in the process of decorin endocytosis. In order to assign the role of the core protein and the GAGs in the internalization and degradation process of decorin, inhibited by RAP, we evaluated the role of the core, the GAGs and both together in the uptake and degradation of [35S]decorin. Fig. 3 shows that the whole unlabeled decorin molecule inhibited both the clearance and degradation of [35S]decorin to the same extent as RAP. The core alone, as well as chondroitin sulfate, was less effective in inhibiting the clearance and degradation. Interestingly, when added together, the inhibition was similar to that observed using the whole decorin, suggesting that both moieties participate in the interaction with LRP and in its subsequent degradation.

Myoblasts Express LRP That Interacts with Decorin—We next evaluated whether C2C12 myoblasts expressed LRP by carrying out RT-PCR assays for LRP using mRNA isolated from C2C12 myoblasts and from U87 cells as a positive control, given that in these cells, the trafficking and function of LRP have been well characterized (53). C2C12 myoblasts were seen to express LRP mRNA (Fig. 4A) as well as the protein, after performing Western blots (Fig. 4B). Finally, we tested for the presence of LRP by indirect immunofluorescence using a confocal microscope, using a polyclonal antibody against human LRP. As seen in Fig. 4C, C2C12 myoblasts were positive for LRP, with the majority of receptors being intracellular, reflecting a high endocytic activity. These results clearly demonstrated the expression of LRP in C2C12 myoblasts. Interestingly, we also found that LRP protein levels diminished during skeletal muscle differentiation, as seen by Western blotting (Fig. 4D).

To evaluate the possible interaction between decorin and LRP at the molecular level, myoblasts were incubated with 125I-labeled decorin core protein at 4 °C to avoid endocytosis, and the cells were lysed and immunoprecipitated with antibodies against the extracellular domain of LRP. After separation by SDS-PAGE, autoradiography revealed that decorin co-immunoprecipitated with the LRP-antibody complexes (Fig. 5A). The same figure shows that this interaction was inhibited by the addition of excess unlabeled decorin core protein, full-length decorin isolated from cartilage, and GST-RAP, whereas GST alone had no effect. To analyze the direct interaction between decorin core protein and LRP at the cell surface, C2C12 myoblasts were incubated with [125I]decorin core protein at 4 °C for 3 h, after which excess labeled decorin was removed, a cross-linker agent (DSS) was added, and cell lysates were immunoprecipitated against LRP. SDS-PAGE followed by autoradiography (Fig. 5B) showed that decorin core protein interacted with LRP and migrated as a high molecular weight complex. To determine whether RAP was able to affect the formation of this complex, the same cross-linking experiment was carried out in the presence of GST-RAP. As seen in Fig. 5C, GST-RAP inhibited the formation of the high molecular weight complex, in contrast to controls or the incubation of myoblasts with GST alone (seen in Fig. 5A). Together, these results not only show that myoblasts express LRP but also show that decorin specifically interacts with this receptor.


Figure 5
View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 5.
[125I]decorin core protein binds to LRP in C2C12 cells. A, myoblasts were incubated with [125I]decorin core protein at 4 °C for 3 h in the absence (Control) or presence of unlabeled decorin core protein (Dcn core), full-length decorin (Dcn full), GST, or GST-RAP. Cells were washed and lysed, and extracts were immunoprecipitated using anti-LRP antibodies. Radiolabeled immunoprecipitated proteins were visualized by phosphorimaging (upper panel), and total LRP protein levels were determined by Western blotting (lower panel). B, myoblasts were incubated with [125I]decorin core protein at 4 °C for 3 h and then cross-linked, followed by immunoprecipitation with anti-LRP antibodies. Radiolabeled immunoprecipitated proteins were visualized under a PhosphorImager. C, myoblasts were incubated with [125I]decorin core protein at 4 °C for 3 h, in the absence (Ctrl) or presence of either GST or GST-RAP. After cross-linking, cell extracts were electrophoretically separated by SDS-PAGE. Gels were stained, dried, and exposed under a PhosphorImager to detect radio-labeled proteins (upper panel). Total protein levels are shown by Coomassie Blue staining (lower panel).

 
Specific Inhibition of LRP Protein Synthesis Eliminates the Interaction, Endocytosis, and Degradation of Decorin—To focus on the functional relationship between decorin and LRP, C2C12 myoblasts were transfected with a specific siRNA for LRP. Fig. 6A shows that LRP expression was strongly inhibited by LRP siRNA, as has been previously reported (44), whereas transfection with siRNA-Control had no effect on LRP levels in these cells. Under these experimental conditions, the complex previously formed between [125I]decorin core protein and LRP, did not arise, as indicated in Fig. 6B. Given that these results provided strong evidence for the formation of a decorin-LRP complex, we next investigated whether inhibiting LRP synthesis would have a functional impact on decorin endocytosis and degradation. Fig. 7A shows that strong inhibition of [35S]decorin clearance occurred in myoblasts transfected with LRP siRNA, in contrast to cells transfected with siRNA-Control. Moreover, the degradation of [35S]decorin was also strongly inhibited by the lack of LRP expression (Fig. 7B). These results all point to LRP as the endocytic receptor for decorin in C2C12 myoblasts.

CHO Cells Rely on LRP Expression in Order to Degrade Decorin Core Protein—To confirm the results obtained in myoblasts, whereby decorin endocytosis and degradation were inhibited by the absence of endogenous LRP resulting from siRNA techniques, we evaluated the ability of a CHO cell line, devoid of LRP expression, to degrade decorin core protein. Fig. 8A shows that, unlike LRP-null CHO cells, wild type CHO-K1 cells were able to degrade [125I]decorin core protein and that this process was strongly inhibited by GST-RAP. As a comparison, the values of core protein degradation and inhibition by GST-RAP are shown for C2C12 myoblasts as well. In order to characterize the LRP ligand-binding domains involved in decorin binding, we transfected LRP-null CHO cells with the cDNA encoding for LRP mini-receptors. These contain, at the ectodomains, the second (mLRP2) or the fourth (mLRP4) ligand-binding domains of LRP and all of the other receptor regions (namely the transmembrane and cytoplasmic domains). As reported for other ligands (43), we found that both minireceptors were able to mediate decorin internalization and degradation in CHO cells (Fig. 8B). These results unequivocally confirm that LRP is an endocytic receptor for decorin and that its ability to recognize decorin involves at least two of its ligand-binding domains.


Figure 6
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 6.
Decorin binding to LRP decreases in myoblasts treated with LRP siRNA. A, myoblasts were transfected with a 75 nM concentration of either control siRNA (si-Ctrl) or LRP siRNA (si-LRP), and control experiments without siRNA (Lipo) were also included (44). Western blotting for LRP was performed in cell extracts, 72 h after transfection. Tubulin is shown as a protein loading control. B, cross-linking assays were carried out in myoblasts incubated with [125I]decorin core protein at 4 °C for 3 h. Following incubation, cells were washed and treated with the cross-linker DSS, extracts were separated by SDS-PAGE, and gels were dried and exposed by phosphorimaging (upper panel). Gel staining with Coomassie Blue is shown in the lower panel.

 


Figure 7
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 7.
Decorin endocytosis and degradation decrease in myoblasts treated with LRP siRNA. As for Fig. 6, myoblasts were transfected with a 75 nM concentration of either control siRNA (si-Ctrl) or LRP siRNA (si-LRP), including control experiments without siRNA (Lipo) (44). 72 h after transfection, cells were incubated with [35S]decorin at 37 °C for 3 h and then analyzed to determine endocytosis (A) and degradation of [35S]decorin (B), as for Fig. 1. Endocytosis is expressed as the clearance of [35S]decorin (volume/mg of protein/h) Percentage degradation values were normalized against control cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this paper, we show for the first time that the multiligand receptor LRP functions as the endocytic receptor for decorin. To sustain this fact, we examine the inhibition of decorin endocytosis and degradation by specifically abolishing the expression of LRP using siRNA and by precluding decorin uptake using RAP, an inhibitor of the interaction between LRP and several ligands (20). Furthermore, experiments using cells that do not express LRP showed these to be unable to take up and degrade decorin. LRP was also found to interact with decorin through the proteoglycan's core protein. Thus, either the whole proteoglycan molecule or its core protein were specifically endocytosed and degraded, and both processes were inhibited by RAP or depletion of LRP using siRNA. We also show that the endocytosis of decorin resulted from a direct interaction between LRP and decorin. Evidence for this was provided by cross-linking assays using radiolabeled decorin and by co-immunoprecipitating LRP with labeled decorin, noting again that both reactions were inhibited by RAP. The interaction between decorin and LRP was deemed specific, since in the presence of exogenous competitors, namely the decorin core protein or the full molecule, the co-immunoprecipitation of LRP and decorin was reduced. The LRP-mediated clearance and degradation processes of sulfated decorin was partially blocked with chondroitin sulfate alone or the core protein of decorin. However, the inhibitory effect of both elements was additive, suggesting that the core and the GAGs moieties have a role in the interaction with LRP.

Experiments measuring the kinetics of decorin internalization and degradation suggested that lysosomes were likely to be involved in these processes, given that the lysosomal inhibitor chloroquine inhibited the degradation of radiolabeled decorin core protein by ~90%. The related proteoglycan biglycan was also able to inhibit decorin degradation, confirming previous findings (54) and suggesting that biglycan could also be endocytosed by LRP. The endocytosis of decorin was first described by Kresse's group (16, 55), although the receptor required for decorin binding and subsequent endocytosis was not identified. A protein of 51 kDa, present in endosomes and at the plasma membrane, has been suggested to act as the decorin receptor due to its high affinity for the decorin core protein (18, 55, 56). However, no data have shown this protein to have a functional role in the uptake and degradation of decorin, as it was clearly shown for LRP in this work. Although our results clearly demonstrate that decorin is endocytosed via LRP and subsequently degraded, a possible role for the 51-kDa protein in these processes cannot be discarded.


Figure 8
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 8.
CHO cells expressing endogenous or recombinant LRP endocytose and degrade decorin. A, LRP-null CHO cells (gray bar) were assayed for [125I]decorin core protein degradation. LRP-expressing C2C12 cells (white bars) and CHO-K1 (wild type) cells (black bars), used as controls, were submitted to competition assays with GST-RAP and GST. B, LRP-null CHO cells, expressing minireceptors containing the second (mLRP2) or fourth (mLRP4) LRP ligand-binding domains, were assayed for [35S]decorin endocytosis and degradation in the absence (control, black bars) or presence of either full-length decorin (Dcn full, light gray bars) or GST-RAP (dark gray bars). CHO-K1 cells and LRP-null CHO cells were used as controls. As before, endocytosis is expressed as the clearance of [35S]decorin (volume/mg of protein/h), and degradation values, shown as percentages, were normalized against controls, in this case CHO-K1 cells, with the corresponding error bars.

 
On the other hand, the endocytic receptor of decorin has also been suggested to contain a binding site for heparin/heparan sulfate, after observation of the inhibition of decorin endocytosis in the presence of heparin (52, 57). Several LRP ligands, such as factor IXa and factor VIII complexed with van Willebrand factor, bind and are also properly presented to the receptor by heparan sulfate proteoglycans (58). This could also be the case for decorin, since its endocytosis and degradation were inhibited by both RAP and heparin. LRP also binds and endocytoses several ECM molecules, including fibronectin (24), thrombospondin (21-23), plasminogen activators (27, 59), and matrix metalloproteinases (27), thereby regulating their bioavailability and functions. These processes involve, in many cases, the activation of signal transduction pathways related to different physiological roles. Thus, LRP participates in events such as cell migration (30, 60), myofibroblast differentiation (33), growth inhibition (61), lipoprotein catabolism (62), angiogenesis, and metastasis (19, 27) among others. Therefore, it is appealing to study the potential role of LRP in modulating the bioavailability and consequent signaling of decorin specifically during myogenesis (14). Here we show that upon differentiation of C2C12 myoblasts, LRP expression decreased, which would have functional implications for several processes, including ECM formation and composition, essential processes that are regulated during differentiation (64). The regulation of LRP expression is complex and depends on the cellular context. For example, both the overexpression and down-regulation of LRP have been observed in different cancers (65, 66). In adipocytes, LRP is up-regulated by peroxisome proliferator-activated receptor {gamma} (67). In PC12 cells, TGF-beta2 increases mRNA and protein expression of LRP (68), and in retinal pigment epithelial cells, LRP-1 mRNA expression is strongly increased upon cell stimulation with TGF-beta1, TGF-beta2, or vascular endothelial growth factor whereas platelet-derived growth factor and fibroblast growth factor type 2 elicited only minor effects insulin treatment (41). Consequently, it would be interesting to characterize the regulatory elements involved in the decrease of LRP expression reported here during skeletal muscle differentiation.

As already mentioned, decorin is also involved in several cellular processes, such as cell adhesion, migration, proliferation, and signaling (for a review see Ref. 71). Therefore, the synthesis and degradation of decorin and accessory molecules required during such processes must be highly regulated. In skeletal muscle, we have previously shown that decorin synthesis is low in myoblasts yet increases during skeletal muscle differentiation (51). This observation is concordant with the requirements of a functional and organized ECM in order for skeletal muscle differentiation to proceed successfully (64, 72). It is well known that decorin interacts with many ECM constituents, the expression of which is also up-regulated during myogenesis, including collagen type I, II, and IV (73, 74) and fibronectin (75), and is present in the correctly organized ECM of skeletal muscle (76, 77). Moreover, decorin is known to induce growth arrest and retard the growth of a variety of tumor cells (78, 79). This growth arrest is associated with an induction of p21, a potent inhibitor of cyclin-dependent kinase activity (80). It has also been suggested that the epidermal growth factor receptor could be involved, through mobilization of intracellular Ca2+, in a possible mechanism by which decorin causes growth suppression (81). Myoblasts are the source of myogenic cells for the formation of skeletal muscle fibers during muscle development and regeneration (82, 83). The number of skeletal muscle pre-cursors or satellite cells is generally quite small, and an active process of myoblast proliferation precedes the formation and repair of injured muscle (84). One can therefore speculate that the active clearance of decorin by LRP from the myoblast conditioned medium, described in this paper, could result in a loss of decorin inhibition upon myoblast proliferation during this stage of differentiation. Since LRP protein levels decrease during skeletal muscle differentiation, it is likely that when proliferation ceases, decorin is partially if not totally incorporated into the ECM. Interestingly, decorin also binds TGF-beta, a strong inhibitor of muscle formation (85), and we have data suggesting that decorin and biglycan modulate the bioavailability of TGF-beta for its transducing receptors by sequestering TGF-beta to the ECM. Indeed, the accumulation of decorin and biglycan at the ECM has a strong inhibitory effect on the binding of TGF-beta to its transducing receptors and its subsequent signaling activity.5

The binding of growth factors to proteoglycans and the consequent modulation of growth factor activities represent an important conceptual advance in the field. In myoblasts, we have shown that the expression of the plasma membrane-associated proteoglycans syndecan-1 and -3 is not only down-regulated during skeletal muscle differentiation (63, 86) but is also critical in order to present fibroblast growth factor type 2 to its transducing receptors and thereby modulate myogenesis (47, 63). We and others have shown that decorin can stimulate TGF-beta-dependent signaling in osteoblasts and nondifferentiated myoblasts (14, 40), and it has been suggested that decorin might interact with certain cell surface proteins or receptors as a means of presenting TGF-beta to its transducing receptors. The fact that LRP binds decorin in myoblasts therefore points to this receptor as a possible candidate for the modulation of TGF-beta activity by decorin, observed previously (14).


   FOOTNOTES
 
* This work was supported in part FONDAP-Biomedicine Grants 13980001, MDA 3790, and FONDECYT 1020726. The Millennium Institute for Fundamental and Applied Biology is financed in part by the Ministerio de Planificación y Cooperación (Chile). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by an International Research Scholar grant from the Howard Hughes Medical Institute. Back

2 Supported by DIPUC and CONICYT Fellowship AT-24050108. Back

3 To whom correspondence should be addressed. Tel.: 56-2-6862112; Fax: 56-2-2229995; E-mail: mmarzolo{at}bio.puc.cl.

4 The abbreviations used are: GAG, glycosaminoglycan; Adv-Dcn, adenovirus containing full-length human decorin; DSS, disuccinimidylsuberate; ECM, extracellular matrix; FBS, fetal bovine serum; LDL, low density lipoprotein; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; siRNA, short interfering RNA; TGF-beta, transforming growth factor type beta; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; RT, reverse transcription. Back

5 Droguett, R., Cabello-Verrugio, C., Riquelme, C., and Brandan, E. (2006) Matrix Biol. 25, 332-341. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Guojun Bu (Washington University School of Medicine, St. Louis, MO) for providing the plasmid encoding mLRP2 and rabbit anti-LRP antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Scott, P. G., McEwan, P. A., Dodd, C. M., Bergmann, E. M., Bishop, P. N., and Bella, J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15633-15638[Abstract/Free Full Text]
  2. Scott, P. G., Grossmann, J. G., Dodd, C. M., Sheehan, J. K., and Bishop, P. N. (2003) J. Biol. Chem. 21, 21
  3. Reed, C. C., and Iozzo, R. V. (2002) Glycoconj. J. 19, 249-255[CrossRef][Medline] [Order article via Infotrieve]
  4. Merle, B., Durussel, L., Delmas, P., and Clezardin, P. (1999) J. Cell. Biochem. 75, 538-546[CrossRef][Medline] [Order article via Infotrieve]
  5. Merle, B., Malaval, L., Lawler, J., Delmas, P., and Clezardin, P. (1997) J. Cell. Biochem. 67, 75-83
  6. Yamaguchi, Y., Mann, D., and Ruoslahti, E. (1990) Nature 346, 281-284[CrossRef][Medline] [Order article via Infotrieve]
  7. Schonherr, E., Sunderkotter, C., Iozzo, R. V., and Schaefer, L. (2005) J. Biol. Chem. 280, 15767-15772[Abstract/Free Full Text]
  8. Tufvesson, E., and Westergren-Thorsson, G. (2002) FEBS Lett. 530, 124-128[CrossRef][Medline] [Order article via Infotrieve]
  9. Biglari, A., Bataille, D., Naumann, U., Weller, M., Zirger, J., Castro, M. G., and Lowenstein, P. R. (2004) Cancer Gene Ther. 11, 721-732[CrossRef][Medline] [Order article via Infotrieve]
  10. Stander, M., Naumann, U., Dumitrescu, L., Heneka, M., Loschmann, P., Gulbins, E., Dichgans, J., and Weller, M. (1998) Gene Ther. 5, 1187-1194[CrossRef][Medline] [Order article via Infotrieve]
  11. Grant, D. S., Yenisey, C., Rose, R. W., Tootell, M., Santra, M., and Iozzo, R. V. (2002) Oncogene 21, 4765-4777[CrossRef][Medline] [Order article via Infotrieve]
  12. Reed, C. C., Gauldie, J., and Iozzo, R. V. (2002) Oncogene 21, 3688-3695[CrossRef][Medline] [Order article via Infotrieve]
  13. Moscatello, D. K., Santra, M., Mann, D. M., McQuillan, D. J., Wong, A. J., and Iozzo, R. V. (1998) J. Clin. Invest. 15, 406-412
  14. Riquelme, C., Larrain, J., Schonherr, E., Henriquez, J. P., Kresse, H., and Brandan, E. (2001) J. Biol. Chem. 276, 3589-3596[Abstract/Free Full Text]
  15. Schmidt, G., Hausser, H., and Kresse, H. (1990) Biochem. J. 266, 591-595[Medline] [Order article via Infotrieve]
  16. Hausser, H., Ober, B., Quentin-Hoffmann, E., Schmidt, B., and Kresse, H. (1992) J. Biol. Chem. 267, 11559-11564[Abstract/Free Full Text]
  17. Truppe, W., and Kresse, H. (1978) Eur. J. Biochem. 85, 351-356[Medline] [Order article via Infotrieve]
  18. Hausser, H., Wedekind, P., Sperber, T., Peters, R., Hasilik, A., and Kresse, H. (1996) Eur. J. Cell Biol. 71, 325-331[Medline] [Order article via Infotrieve]
  19. Herz, J., and Strickland, D. K. (2001) J. Clin. Invest. 108, 779-784[CrossRef][Medline] [Order article via Infotrieve]
  20. Bu, G., and Marzolo, M. P. (2000) Trends Cardiovasc. Med. 10, 148-155[CrossRef][Medline] [Order article via Infotrieve]
  21. Godyna, S., Liau, G., Popa, I., Stefansson, S., and Argraves, W. S. (1995) J. Cell Biol. 129, 1403-1410[Abstract/Free Full Text]
  22. Mikhailenko, I., Kounnas, M. Z., and Strickland, D. K. (1995) J. Biol. Chem. 270, 9543-9549[Abstract/Free Full Text]
  23. Chen, H., Sottile, J., Strickland, D. K., and Mosher, D. F. (1996) Biochem. J. 318, 959-963
  24. Salicioni, A. M., Mizelle, K. S., Loukinova, E., Mikhailenko, I., Strickland, D. K., and Gonias, S. L. (2002) J. Biol. Chem. 277, 16160-16166[Abstract/Free Full Text]
  25. Herz, J., Clouthier, D. E., and Hammer, R. E. (1992) Cell 71, 411-421[CrossRef][Medline] [Order article via Infotrieve]
  26. Bu, G., Williams, S., Strickland, D. K., and Schwartz, A. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7427-7431[Abstract/Free Full Text]
  27. Yang, Z., Strickland, D. K., and Bornstein, P. (2001) J. Biol. Chem. 276, 8403-8408[Abstract/Free Full Text]
  28. Segarini, P. R., Nesbitt, J. E., Li, D., Hays, L. G., Yates, J. R., III, and Carmichael, D. F. (2001) J. Biol. Chem. 276, 40659-40667[Abstract/Free Full Text]
  29. Gonias, S. L., Wu, L., and Salicioni, A. M. (2004) Thromb. Haemost. 91, 1056-1064[Medline] [Order article via Infotrieve]
  30. Orr, A. W., Elzie, C. A., Kucik, D. F., and Murphy-Ullrich, J. E. (2003) J. Cell Sci. 116, 2917-2927[Abstract/Free Full Text]
  31. Boucher, P., Liu, P., Gotthardt, M., Hiesberger, T., Anderson, R. G., and Herz, J. (2002) J. Biol. Chem. 277, 15507-15513[Abstract/Free Full Text]
  32. Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini, M. M., Loukinov, D., Ulery, P. G., Mikhailenko, I., Lawrence, D. A., and Strickland, D. K. (2002) J. Biol. Chem. 277, 15499-15506[Abstract/Free Full Text]
  33. Yang, M., Huang, H., Li, J., Li, D., and Wang, H. (2004) FASEB J. 18, 1920-1921[Abstract/Free Full Text]
  34. Huang, S. S., Leal, S. M., Chen, C. L., Liu, I. H., and Huang, J. S. (2004) FASEB J. 18, 1719-1721[Abstract/Free Full Text]
  35. Tseng, W. F., Huang, S. S., and Huang, J. S. (2004) FEBS Lett. 562, 71-78[CrossRef][Medline] [Order article via Infotrieve]
  36. Schonherr, E., Broszat, M., Brandan, E., Bruckner, P., and Kresse, H. (1998) Arch. Biochem. Biophys. 355, 241-248[CrossRef][Medline] [Order article via Infotrieve]
  37. Schmidt, G., Hausser, H., and Kresse, H. (1991) Biochem. J. 280, 411-414
  38. Winnemoller, M., Schon, P., Vischer, P., and Kresse, H. (1992) Eur. J. Cell Biol. 59, 47-55[Medline] [Order article via Infotrieve]
  39. Nili, N., Cheema, A. N., Giordano, F. J., Barolet, A. W., Babaei, S., Hickey, R., Eskandarian, M. R., Smeets, M., Butany, J., Pasterkamp, G., and Strauss, B. H. (2003) Am. J. Pathol. 163, 869-878[Abstract/Free Full Text]
  40. Takeuchi, Y., Kodama, Y., and Matsumoto, T. (1994) J. Biol. Chem. 269, 32634-32638[Abstract/Free Full Text]
  41. Boucher, P., Ducluzeau, P. H., Davelu, P., Andreelli, F., Vallier, P., Riou, J. P., Laville, M., and Vidal, H. (2002) Biochim. Biophys. Acta 1588, 226-231[Medline] [Order article via Infotrieve]
  42. Marzolo, M. P., Yuseff, M. I., Retamal, C., Donoso, M., Ezquer, F., Farfan, P., Li, Y., and Bu, G. (2003) Traffic 4, 273-288[Medline] [Order article via Infotrieve]
  43. Obermoeller-McCormick, L. M., Li, Y., Osaka, H., FitzGerald, D. J., Schwartz, A. L., and Bu, G. (2001) J. Cell Sci. 114, 899-908[Abstract]
  44. Li, Y., Lu, W., and Bu, G. (2003) FEBS Lett. 555, 346-350[CrossRef][Medline] [Order article via Infotrieve]
  45. Cuitino, L., Matute, R., Retamal, C., Bu, G., Inestrosa, N. C., and Marzolo, M. P. (2005) Traffic 6, 820-838[CrossRef][Medline] [Order article via Infotrieve]
  46. Yaffe, D., and Saxel, O. (1977) Nature 270, 725-727[CrossRef][Medline] [Order article via Infotrieve]
  47. Larrain, J., Carey, D. J., and Brandan, E. (1998) J. Biol. Chem. 273, 32288-32296[Abstract/Free Full Text]
  48. Li, Y., Marzolo, M. P., van Kerkhof, P., Strous, G. J., and Bu, G. (2000) J. Biol. Chem. 275, 17187-17194[Abstract/Free Full Text]
  49. Lopez-Casillas, F., Riquelme, C., Perez-Kato, Y., Ponce-Castaneda, M. V., Osses, N., Esparza-Lopez, J., Gonzalez-Nunez, G., Cabello-Verrugio, C., Mendoza, V., Troncoso, V., and Brandan, E. (2003) J. Biol. Chem. 278, 382-390[Abstract/Free Full Text]
  50. Obermoeller, L. M., Chen, Z., Schwartz, A. L., and Bu, G. (1998) J. Biol. Chem. 273, 22374-22381[Abstract/Free Full Text]
  51. Brandan, E., Fuentes, M. E., and Andrade, W. (1991) Eur. J. Cell Biol. 55, 209-216[Medline] [Order article via Infotrieve]
  52. Hausser, H., Witt, O., and Kresse, H. (1993) Exp. Cell Res. 208, 398-406[CrossRef][Medline] [Order article via Infotrieve]
  53. Bu, G., Maksymovitch, E. A., Geuze, H., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 29874-29882[Abstract/Free Full Text]
  54. Hausser, H., Schonherr, E., Muller, M., Liszio, C., Bin, Z., Fisher, L., and Kresse, H. (1998) Arch. Biochem. Biophys. 349, 363-370[CrossRef][Medline] [Order article via Infotrieve]
  55. Hausser, H., and Kresse, H. (1991) J. Cell Biol. 114, 45-52[Abstract/Free Full Text]
  56. Hausser, H., Hoppe, W., Rauch, U., and Kresse, H. (1989) Biochem. J. 263, 137-142[Medline] [Order article via Infotrieve]
  57. Hausser, H., and Kresse, H. (1999) Biochem. J. 344, 827-835
  58. Nykjaer, A., and Willnow, T. E. (2002) Trends Cell Biol. 12, 273-280[CrossRef][Medline] [Order article via Infotrieve]
  59. Warshawsky, I., Herz, J., Broze, G. J., Jr., and Schwartz, A. L. (1996) J. Biol. Chem. 271, 25873-25879[Abstract/Free Full Text]
  60. Webb, D. J., Nguyen, D. H., and Gonias, S. L. (2000) J. Cell Sci. 113, 123-134[Abstract]
  61. Huang, S. S., Ling, T. Y., Tseng, W. F., Huang, Y. H., Tang, F. M., Leal, S. M., and Huang, J. S. (2003) FASEB J. 17, 2068-2081[Abstract/Free Full Text]
  62. Wilsie, L. C., and Orlando, R. A. (2003) J. Biol. Chem. 278, 15758-15764[Abstract/Free Full Text]
  63. Fuentealba, L., Carey, D. J., and Brandan, E. (1999) J. Biol. Chem. 274, 37876-37884[Abstract/Free Full Text]
  64. Osses, N., and Brandan, E. (2002) Am. J. Physiol. 282, C383-C394
  65. Desrosiers, R. R., Rivard, M. E., Grundy, P. E., and Annabi, B. (2006) Pediatr. Blood Cancer 46, 40-49[CrossRef][Medline] [Order article via Infotrieve]
  66. Benes, P., Jurajda, M., Zaloudik, J., Izakovicova-Holla, L., and Vacha, J. (2003) Breast Cancer Res. 5, R77-R81[CrossRef][Medline] [Order article via Infotrieve]
  67. Gauthier, A., Vassiliou, G., Benoist, F., and McPherson, R. (2003) J. Biol. Chem. 278, 11945-11953[Abstract/Free Full Text]
  68. Harris-White, M. E., and Frautschy, S. A. (2005) Curr. Drug Targets CNS Neurol. Disord. 4, 469-480[CrossRef][Medline] [Order article via Infotrieve]
  69. Hollborn, M., Birkenmeier, G., Saalbach, A., Iandiev, I., Reichenbach, A., Wiedemann, P., and Kohen, L. (2004) Invest. Ophthalmol. Vis. Sci. 45, 2033-2038[Abstract/Free Full Text]
  70. Llorente-Cortes, V., Otero-Vinas, M., Sanchez, S., Rodriguez, C., and Badimon, L. (2002) Circulation 106, 3104-3110[Abstract/Free Full Text]
  71. Iozzo, R. V. (1999) J. Biol. Chem. 274, 18843-18846[Free Full Text]
  72. Melo, F., Carey, D. J., and Brandan, E. (1996) J. Cell. Biochem. 62, 227-239[CrossRef][Medline] [Order article via Infotrieve]
  73. Bidanset, D. J., Guidry, C., Rosenberg, L. C., Choi, H. U., Timpl, R., and Hook, M. (1992) J. Biol. Chem. 267, 5250-5256[Abstract/Free Full Text]
  74. Scott, J. E., and Hughes, E. W. (1986) Connect. Tissue Res. 14, 267-278[Medline] [Order article via Infotrieve]
  75. Asundi, V. K., and Dreher, K. L. (1992) Eur. J. Cell Biol. 59, 314-321[Medline] [Order article via Infotrieve]
  76. Brandan, E., Fuentes, M. E., and Andrade, W. (1992) J. Neurosci. Res. 32, 51-59[CrossRef][Medline] [Order article via Infotrieve]
  77. Caceres, S., Cuellar, C., Casar, J. C., Garrido, J., Schaefer, L., Kresse, H., and Brandan, E. (2000) Eur. J. Cell Biol. 79, 173-181[CrossRef][Medline] [Order article via Infotrieve]
  78. Santra, M., Mann, D. M., Mercer, E. W., Skorski, T., Calabretta, B., and Iozzo, R. V. (1997) J. Clin. Invest. 100, 149-157[Medline] [Order article via Infotrieve]
  79. Santra, M., Skorski, T., Calabretta, B., Lattime, E. C., and Iozzo, R. V. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7016-7020[Abstract/Free Full Text]
  80. De Luca, A., Santra, M., Baldi, A., Giordano, A., and Iozzo, R. V. (1996) J. Biol. Chem. 271, 18961-18965[Abstract/Free Full Text]
  81. Patel, S., Santra, M., McQuillan, D. J., Iozzo, R. V., and Thomas, A. P. (1998) J. Biol. Chem. 273, 3121-3124[Abstract/Free Full Text]
  82. Bischoff, R. (1986) Dev. Biol. 115, 129-139[CrossRef][Medline] [Order article via Infotrieve]
  83. Casar, J. C., McKechnie, B. A., Fallon, J. R., Young, M. F., and Brandan, E. (2004) Dev. Biol. 268, 358-371[CrossRef][Medline] [Order article via Infotrieve]
  84. Mouly, V., Aamiri, A., Bigot, A., Cooper, R. N., Di Donna, S., Furling, D., Gidaro, T., Jacquemin, V., Mamchaoui, K., Negroni, E., Perie, S., Renault, V., Silva-Barbosa, S. D., and Butler-Browne, G. S. (2005) Acta Physiol. Scand. 184, 3-15[CrossRef][Medline] [Order article via Infotrieve]
  85. Florini, J. R., Roberts, A. B., Ewton, D. Z., Falen, S. L., Flanders, K. C., and Sporn, M. B. (1986) J. Biol. Chem. 261, 16509-16513[Abstract/Free Full Text]
  86. Larrain, J., Cizmeci-Smith, G., Troncoso, V., Stahl, R. C., Carey, D. J., and Brandan, E. (1997) J. Biol. Chem. 272, 18418-18424[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
M. Donoso, J. Cancino, J. Lee, P. van Kerkhof, C. Retamal, G. Bu, A. Gonzalez, A. Caceres, and M.-P. Marzolo
Polarized Traffic of LRP1 Involves AP1B and SNX17 Operating on Y-dependent Sorting Motifs in Different Pathways
Mol. Biol. Cell, January 1, 2009; 20(1): 481 - 497.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X. Li, A. Pennisi, and S. Yaccoby
Role of decorin in the antimyeloma effects of osteoblasts
Blood, July 1, 2008; 112(1): 159 - 168.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Cabello-Verrugio and E. Brandan
A Novel Modulatory Mechanism of Transforming Growth Factor-beta Signaling through Decorin and LRP-1
J. Biol. Chem., June 29, 2007; 282(26): 18842 - 18850.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/42/31562    most recent
M602919200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brandan, E.
Right arrow Articles by Marzolo, M.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brandan, E.
Right arrow Articles by Marzolo, M.-P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement