The Low Density Lipoprotein Receptor-related Protein Functions as an Endocytic Receptor for Decorin
2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês
10.1016/s0021-9258(19)84070-x
ISSN1083-351X
AutoresEnrique Brandan, Cláudio Retamal, Claudio Cabello‐Verrugio, Mari ́a-Paz Marzolo,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoDecorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type β 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. Decorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type β 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. 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) 4The 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-β, transforming growth factor type β; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; RT, reverse transcription. 4The 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-β, transforming growth factor type β; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; RT, reverse transcription. chain at its NH2 terminus. A crystal structure for bovine decorin has been published (1Scott P.G. McEwan P.A. Dodd C.M. Bergmann E.M. Bishop P.N. Bella J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15633-15638Crossref PubMed Scopus (179) Google Scholar) that together with earlier x-ray scattering data (2Scott P.G. Grossmann J.G. Dodd C.M. Sheehan J.K. Bishop P.N. J. Biol. Chem. 2003; 21: 21Google Scholar) suggests decorin to be a dimeric protein. Each monomer adopts a curved structure, whereby antiparallel dimerization occurs through the β-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 (3Reed C.C. Iozzo R.V. Glycoconj. J. 2002; 19: 249-255Crossref PubMed Scopus (307) Google Scholar). The interaction of decorin with fibronectin and thrombospondin leads to the inhibition of fibroblast attachment to these substrata (4Merle B. Durussel L. Delmas P. Clezardin P. J. Cell. Biochem. 1999; 75: 538-546Crossref PubMed Scopus (87) Google Scholar, 5Merle B. Malaval L. Lawler J. Delmas P. Clezardin P. J. Cell. Biochem. 1997; 67: 75-83Crossref PubMed Scopus (62) Google Scholar). 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-β (TGF-β) (6Yamaguchi Y. Mann D. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1295) Google Scholar), bind to the insulin-like growth factor-I (7Schonherr E. Sunderkotter C. Iozzo R.V. Schaefer L. J. Biol. 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Oncogene. 2002; 21: 3688-3695Crossref PubMed Scopus (125) Google Scholar). 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 (13Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 15: 406-412Crossref Scopus (245) Google Scholar). 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 (7Schonherr E. Sunderkotter C. Iozzo R.V. Schaefer L. J. Biol. Chem. 2005; 280: 15767-15772Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). On the other hand, reducing decorin levels results in a decreased cell responsiveness to TGF-β, suggesting that decorin is required to activate the TGF-β signaling pathways (14Riquelme C. Larrain J. Schonherr E. Henriquez J.P. Kresse H. Brandan E. J. Biol. Chem. 2001; 276: 3589-3596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). 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 (15Schmidt G. Hausser H. Kresse H. Biochem. J. 1990; 266: 591-595PubMed Google Scholar). Fibroblasts and other cells of mesenchymal origin are known to efficiently internalize decorin by receptor-mediated endocytosis (16Hausser H. Ober B. Quentin-Hoffmann E. Schmidt B. Kresse H. J. Biol. Chem. 1992; 267: 11559-11564Abstract Full Text PDF PubMed Google Scholar, 17Truppe W. Kresse H. Eur. J. Biochem. 1978; 85: 351-356Crossref PubMed Scopus (37) Google Scholar). 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 (18Hausser H. Wedekind P. Sperber T. Peters R. Hasilik A. Kresse H. Eur. J. Cell Biol. 1996; 71: 325-331PubMed Google Scholar). However, no functional evidence is as yet available to support this notion, so that the identity of the decorin receptor remains an open question. 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 (19Herz J. Strickland D.K. J. Clin. Invest. 2001; 108: 779-784Crossref PubMed Scopus (882) Google Scholar). The receptor's folding process in the endoplasmic reticulum requires the participation of the 39-kDa receptor chaperone, RAP (20Bu G. Marzolo M.P. Trends Cardiovasc. Med. 2000; 10: 148-155Crossref PubMed Scopus (55) Google Scholar). 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 (21Godyna S. Liau G. Popa I. Stefansson S. Argraves W.S. J. Cell Biol. 1995; 129: 1403-1410Crossref PubMed Scopus (127) Google Scholar, 22Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 23Chen H. Sottile J. Strickland D.K. Mosher D.F. Biochem. 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Furthermore, LRP regulates signaling cascades by binding ECM molecules, such as fibronectin (29Gonias S.L. Wu L. Salicioni A.M. Thromb. Haemost. 2004; 91: 1056-1064Crossref PubMed Scopus (0) Google Scholar) and thrombospondin (30Orr A.W. Elzie C.A. Kucik D.F. Murphy-Ullrich J.E. J. Cell Sci. 2003; 116: 2917-2927Crossref PubMed Scopus (148) Google Scholar), and growth factors, such as platelet-derived growth factor (31Boucher P. Liu P. Gotthardt M. Hiesberger T. Anderson R.G. Herz J. J. Biol. Chem. 2002; 277: 15507-15513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 32Loukinova E. Ranganathan S. Kuznetsov S. Gorlatova N. Migliorini M.M. Loukinov D. Ulery P.G. Mikhailenko I. Lawrence D.A. Strickland D.K. J. Biol. Chem. 2002; 277: 15499-15506Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), connective tissue growth factor (33Yang M. Huang H. Li J. Li D. Wang H. 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Given the role of decorin in myoblast differentiation (14Riquelme C. Larrain J. Schonherr E. Henriquez J.P. Kresse H. Brandan E. J. Biol. Chem. 2001; 276: 3589-3596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 40Takeuchi Y. Kodama Y. Matsumoto T. J. Biol. Chem. 1994; 269: 32634-32638Abstract Full Text PDF PubMed Google Scholar) and the presence of LRP mRNA in human skeletal muscle cells (41Boucher P. Ducluzeau P.H. Davelu P. Andreelli F. Vallier P. Riou J.P. Laville M. Vidal H. Biochim. Biophys. Acta. 2002; 1588: 226-231Crossref PubMed Scopus (8) Google Scholar), 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. Reagents—Plasmids encoding LRP minireceptors, which include the ligand-binding domains 2 (mLRP2) and 4 (mLRP4) have been described previously (42Marzolo M.P. Yuseff M.I. Retamal C. Donoso M. Ezquer F. Farfan P. Li Y. Bu G. Traffic. 2003; 4: 273-288Crossref PubMed Scopus (73) Google Scholar, 43Obermoeller-McCormick L.M. Li Y. Osaka H. FitzGerald D.J. Schwartz A.L. Bu G. J. Cell Sci. 2001; 114: 899-908Crossref PubMed Google Scholar). Annealed LRP-1-specific siRNA as well as control siRNA were obtained from Ambion (Austin, TX), with LRP siRNA sequences described in Ref. 44Li Y. Lu W. Bu G. FEBS Lett. 2003; 555: 346-350Crossref PubMed Scopus (36) Google Scholar. GST and GST-RAP were produced as described in (45Cuitino L. Matute R. Retamal C. Bu G. Inestrosa N.C. Marzolo M.P. Traffic. 2005; 6: 820-838Crossref PubMed Scopus (58) Google Scholar). Rabbit anti-LRP antibody was kindly provided by Dr. Guojun Bu and used as prescribed by Marzolo et al. (42Marzolo M.P. Yuseff M.I. Retamal C. Donoso M. Ezquer F. Farfan P. Li Y. Bu G. Traffic. 2003; 4: 273-288Crossref PubMed Scopus (73) Google Scholar). Mouse anti-LRP raised against the cytoplasmic domain was purchased from Calbiochem, and mouse anti-α-tubulin was from Sigma. Adenoviral vector containing the full-length cDNA for human decorin (Adv-Dcn) has already been described (14Riquelme C. Larrain J. Schonherr E. Henriquez J.P. Kresse H. Brandan E. J. Biol. Chem. 2001; 276: 3589-3596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). 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) (46Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1559) Google Scholar) was grown and induced to differentiate, as described in Ref. 47Larrain J. Carey D.J. Brandan E. J. Biol. Chem. 1998; 273: 32288-32296Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar. 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) (48Li Y. Marzolo M.P. van Kerkhof P. Strous G.J. Bu G. J. Biol. Chem. 2000; 275: 17187-17194Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) 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 (14Riquelme C. Larrain J. Schonherr E. Henriquez J.P. Kresse H. Brandan E. J. Biol. Chem. 2001; 276: 3589-3596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) 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 (49Lopez-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. Brandan E. J. Biol. Chem. 2003; 278: 382-390Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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 (50Obermoeller L.M. Chen Z. Schwartz A.L. Bu G. J. Biol. Chem. 1998; 273: 22374-22381Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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 (49Lopez-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. Brandan E. J. Biol. Chem. 2003; 278: 382-390Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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 (51Brandan E. Fuentes M.E. Andrade W. Eur. J. Cell Biol. 1991; 55: 209-216PubMed Google Scholar), 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 (50Obermoeller L.M. Chen Z. Schwartz A.L. Bu G. J. Biol. Chem. 1998; 273: 22374-22381Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), 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. 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-α-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 (16Hausser H. Ober B. Quentin-Hoffmann E. Schmidt B. Kresse H. J. Biol. Chem. 1992; 267: 11559-11564Abstract Full Text PDF PubMed Google Scholar). 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. 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. (16Hausser H. Ober B. Quentin-Hoffmann E. Schmidt B. Kresse H. J. Biol. Chem. 1992; 267: 11559-11564Abstract Full Text PDF PubMed Google Scholar). 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 (16Hausser H. Ober B. Quentin-Hoffmann E. Schmidt B. Kresse H. J. Biol. Chem. 1992; 267: 11559-11564Abstract Full Text PDF PubMed Google Scholar). Moreover, it has been previously demonstrated that [35S]decorin endocytosis and degradation decrease in the presence of heparin (52Hausser H. Witt O. Kresse H. Exp. Cell Res. 1993; 208: 398-406Crossref PubMed Scopus (16) Google Scholar). 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. 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 ro
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