Biomedical subjects

W S Argraves

Publications and source records attributed to W S Argraves.

At least 37 records · Page 2Linked to original sources

PubMed1996

Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen.

Fibulin-1 is a component of the extracellular matrix that surrounds vascular smooth muscle. This observation, along with the recent finding that fibulin-1 can bind fibrinogen (J Biol Chem 270:19458, 1995), prompted investigation into the potential role of fibulin-1 as a thrombogenic agent. In perfusion chamber assays, platelets in whole blood under flow conditions attached and spread on surfaces coated with fibulin-1. This adhesion was completely blocked by fibulin-1 antibodies. Platelets free of plasma did not attach to fibulin-1 coated surfaces; however, with the addition of fibrinogen, platelet adhesion to fibulin-1 took place. When detergent extracts of platelets were subjected to fibulin-1-Sepharose affinity chromatography, the integrin alpha IIb beta 3 was selected. Solid phase binding assays using purified components showed that integrin alpha IIb beta 3 could not bind directly to fibulin-1 but in the presence of fibrinogen the integrin bound to fibulin-1-coated surfaces. Monoclonal alpha IIb beta 3 antibodies capable of blocking its interaction with fibrinogen completely blocked platelet adhesion to fibulin-1 in both whole blood perfusion and static adhesion assays. The results show that fibulin-1 can support platelet attachment via a bridge of fibrinogen to the platelet integrin alpha IIb beta 3. When fibroblast monolayers containing extracellular matrix-incorporated fibulin-1 were used as adhesion substrates, platelet adhesion in the presence of fibrinogen could be inhibited by 30% using antibodies to fibulin-1. Following vascular injury, fibulin-1 present in the extracellular matrix of the vessel wall may therefore interact with plasma fibrinogen and promote platelet adhesion, leading to the formation of a platelet plug. Thus, fibulin-1 joins the list of matrix proteins including collagens I and IV and fibronectin that mediate platelet adhesion via a plasma protein bridge. This bridging phenomenon may represent a general mechanism by which platelets interact with exposed subendothelial matrices following vascular injury.

Animals↗

PubMed1996

Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers.

The building of the vessel wall from its cellular and extracellular matrix (ECM) components is a critical event in the development and maturation of the cardiovascular system. However, little is known about the events that occur after the initial vascular network, a nascent endothelium, is established. The proper recruitment of vascular smooth muscle cells (VSMCs) to the endothelium is one such critical event. Although the majority of VSMCs are of mesodermal origin, it is not understood which populations of embryonic cells are capable of following the VSMC differentiation pathway. Previous studies, which have focused on the VSMC component of vessel wall development, have been limited by the use of markers that are not smooth muscle specific, or have focused on events that occur after a multilayered wall has been established. Therefore, the initial goal of this study was to define when overtly identifiable VSMCs were first associated with the vascular endothelium. Monoclonal antibodies (MAbs) were generated from embryonic vessel wall antigens in order to circumvent problems of cell specificity associated with the use of previously available markers to VSMCs. Critical to this study is our MAb, 1E12, which unlike other antibody markers, is smooth muscle specific. Using 1E12, we defined a pattern for recruitment and differentiation of the VSMC component of the descending aorta in stage 12 to stage 20 (Hamburger and Hamilton, 1951) quail embryos. Immunofluorescent labeling of quail embryos with 1E12 and a MAb to smooth muscle alpha-actin (SM alpha A) shows that the first mesodermally derived cells to associate with the aortic endothelium do so at the ventral surface. Recruitment of these cells, which we believe to be primordial VSMCs, proceeds in a ventral to dorsal direction along the aorta and in a radial direction, emanating from the endothelium. Additionally, we have determined the distribution of several ECM proteins, during the initial events of vessel wall development. Our studies show that fibulin-1 is expressed surrounding the primordial VSMCs of the vessel wall before elastin precursors are present and suggest that differential expression of the JB3 antigen (Wunsch et al., 1994) may be indicative of early diversity among embryonic VSMCs.

Animals↗

PubMed1996

Plasminogen activator inhibitor-1 and vitronectin promote the cellular clearance of thrombin by low density lipoprotein receptor-related proteins 1 and 2.

Thrombin is a multifunctional protein that has both proteinase and growth factor-like activities. Its regulation is largely mediated by interaction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha2-macroglobulin (alpha2-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha2-M are all abundant in blood and can inactivate blood-borne thrombin leading to rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin, can efficiently inhibit thrombin in the presence of native vitronectin (VN). In this study, active thrombin was found to be efficiently endocytosed and degraded by cultured pre-type II pneumocyte cells, and both processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in complex with a number of inhibitors was examined, 125I-thrombin-PAI-1 complexes were most efficiently cleared compared to 125I-thrombin in complex with the serpins ATIII, HCII, alpha1-proteinase inhibitor, or d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone. Low density lipoprotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-1, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked 125I-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added 125I-thrombin by cultured cells was found to be enhanced 5-fold by inclusion of wild-type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is unable to bind VN was included. This wild-type PAI-1 enhancement of 125I-thrombin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a novel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP-2. This pathway is potentially important for the regulation of the potent biological activities of thrombin, particularly at sites of vascular injury.

Animals↗

PubMed1996

Cellular internalization and degradation of antithrombin III-thrombin, heparin cofactor II-thrombin, and alpha 1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein.

The inhibition of proteinase activity by members of the serine proteinase inhibitor (serpin) family is a critical regulatory mechanism for a variety of biological processes. Once formed, the serpin enzyme complexes (SECs) are removed from the circulation by a hepatic receptor. The present study suggests that this receptor is very likely the low density lipoprotein receptor-related protein (LRP), a prominent liver receptor. In vitro binding studies revealed that antithrombin III (ATIII)-thrombin, heparin cofactor II (HCII)-thrombin, and alpha1-antitrypsin (alpha1AT)-trypsin bound to purified LRP, and their binding was inhibited by the 39-kDa receptor-associated protein (RAP), an antagonist of LRP-ligand binding activity. In contrast, native or modified forms of the inhibitors were unable to bind to LRP. Mouse embryonic fibroblasts, which express LRP, mediate the cellular internalization leading to degradation of these SECs, while mouse fibroblasts genetically deficient in LRP showed no capacity to internalize and degrade these complexes. SECs were also degraded by HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and chloroquine. The cellular-mediated uptake and degradation was specific for SECs; native or modified forms of the inhibitors were not internalized and degraded. Finally, in vivo clearance studies in rats demonstrated that RAP inhibited the clearance of ATIII-125I-thrombin complexes from the circulation. Together, these results indicate that LRP functions as a liver receptor responsible for the plasma clearance of SECs.

Animals↗

PubMed1996

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.

Adenocarcinoma↗

PubMed1996

Fibulin-1, vitronectin, and fibronectin expression during avian cardiac valve and septa development.

BACKGROUND: Extracellular matrix (ECM) proteins have been implicated as mediators of events important to valvuloseptal development (reviewed by Little and Rongish, Experentia, 51:873-882, 1995). The aim of this study was to identify connective tissue ECM proteins present at sites of valvuloseptal morphogenesis, and to determine how their patterns of expression change during the developmental process. METHODS: Immunofluorescence microscopy was used to examine the distribution of fibulin-1, vitronectin, and fibronectin in the embryonic chicken heart over a broad developmental time frame (Hamburger and Hamilton stages 14 to 44), emphasizing stages that illustrate endocardial cushion formation, growth, fusion, and development into valvuloseptal components. RESULTS AND CONCLUSIONS: Fibulin-1 immunolabeling was concentrated in endocardial cushions, notably at boundaries with the myocardium, during stages when the cushions are differentiating into valvular and septal components. Fibulin-1 was detected in the endocardial cushions prior to their seeding with cushion cells, but became undetectable by early midgestation. Vitronectin expression was similar to fibulin-1, but less restricted in its distribution. Vitronectin was observed before endocardial cushion cell migration commenced and persisted until the formation of prevalvular structures (early midgestation) in the atrioventricular cushions. Vitronectin remained detectable in the semilunar valves until late midgestation. Fibronectin was present in the endocardial cushion region and in portions of the endocardium and myocardium throughout the stages presented. Our data suggests that the ECM of the endocardial cushions undergoes remodelling in a regionally and temporally specific manner which corresponds with morphogenetic changes during valvuloseptal development.

Animals↗

PubMed1996

Low density lipoprotein receptor-related protein-2 expression in efferent duct and epididymal epithelia: evidence in rats for its in vivo role in endocytosis of apolipoprotein J/clusterin.

Apolipoprotein J/clusterin/sulfated glycoprotein-2 (apo J) disassociates from spermatozoa and is endocytosed by epithelial cells lining the efferent ducts and epididymis. The low density lipoprotein receptor-related protein-2/megalin (LRP-2) has been shown to bind to apo J and mediates its endocytosis and lysosomal degradation in cultured cells. In this study, immunocytological techniques were used to localize LRP-2 in rat efferent ducts and epididymis and to determine whether its expression correlated with those epithelial cells involved in apo J endocytosis. Pronounced LRP-2 immunochemical staining was observed on the apical surfaces of epithelial cells lining the efferent ducts and in the intermediate zone, proximal caput, and corpus and cauda regions of the epididymis. Single immunogold labeling at the electron microscopic level showed LRP-2 to be present within coated pits, endocytic vesicles, and early endosomes of the nonciliated cells of the efferent ducts and the principal cells of the epididymis. In efferent ducts, double immunogold labeling showed both LRP-2 and apo J to be present in endocytic compartments including coated pits, endocytic vesicles, and early endosomes of nonciliated cells. However, while apo J was detected in late endosomes and lysosomes of nonciliated cells, LRP-2 was not. Apical tubules, possibly emerging from late endosomes, contained labeling for LRP-2 but not for apo J. Ciliated cells lying adjacent to nonciliated cells displayed no labeling for either LRP-2 or apo J. These results are consistent with the possibility that LRP-2 serves as an endocytic receptor for apo J in vivo and that after endocytosis the LRP-2 is recycled back to the cell surface while apo J is delivered to the lysosomes for degradation. To provide additional evidence implicating LRP-2 in apo J endocytosis, a receptor-associated protein (RAP), an antagonist of apo J binding to LRP-2, was injected into the efferent duct lumen. Subsequent immunocytological analysis of the efferent duct showed that the RAP treatment abolished the endocytosis of apo J by the nonciliated cells. Taken together, these data indicate that LRP-2 is a likely mediator of apo J endocytosis by the nonciliated efferent duct cells.

Animals↗

PubMed1995

Glycoprotein 330/low density lipoprotein receptor-related protein-2 mediates endocytosis of low density lipoproteins via interaction with apolipoprotein B100.

The ability of glycoprotein 330/low density lipoprotein receptor-related protein-2 (LRP-2) to function as a lipoprotein receptor was investigated using cultured mouse F9 teratocarcinoma cells. Treatment with retinoic acid and dibutyryl cyclic AMP, which induces F9 cells to differentiate into endoderm-like cells, produced a 50-fold increase in the expression of LRP-2. Levels of the other members of the low density lipoprotein (LDL) receptor (LDLR) family, including LDLR, the very low density lipoprotein receptor, and LRP-1, were reduced. When LDL catabolism was examined in these cells, it was found that the treated cells endocytosed and degraded at 10-fold higher levels than untreated cells. The increased LDL uptake coincided with increased LRP-2 activity of the treated cells, as measured by uptake of both 125I-labeled monoclonal LRP-2 antibody and the LRP-2 ligand prourokinase. The ability of LDL to bind to LRP-2 was demonstrated by solid-phase binding assays. This binding was inhibitable by LRP-2 antibodies, receptor-associated protein (the antagonist of ligand binding for all members of the LDLR family), or antibodies to apoB100, the major apolipoprotein component of LDL. In cell assays, LRP-2 antibodies blocked the elevated 125I-LDL internalization and degradation observed in the retinoic acid/dibutyryl cyclic AMP-treated F9 cells. A low level of LDL endocytosis existed that was likely mediated by LDLR since it could not be inhibited by LRP-2 antibodies, but was inhibited by excess LDL, receptor-associated protein, or apoB100 antibody. The results indicate that LRP-2 can function to mediate cellular endocytosis of LDL, leading to its degradation. LRP-2 represents the second member of the LDLR family identified as functioning in the catabolism of LDL.

Animals↗

PubMed1995

The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis.

The fibulins are an emerging family of extracellular matrix and blood proteins presently having two members designated fibulin-1 and -2. Fibulin-1 is the predominant fibulin in blood, present at a concentration of 30-40 micrograms/ml (approximately 1000-fold higher than fibulin-2). During the course of isolating fibulin-1 from plasma by immunoaffinity chromatography, a 340-kDa polypeptide was consistently found to co-purify. This protein was identified as fibrinogen (Fg) based on its electrophoretic behavior and reactivity with Fg monoclonal antibodies. Radioiodinated fibulin-1 was shown to bind to Fg transferred onto nitrocellulose filters after SDS-polyacrylamide gel electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound to Fg (and fibrin) adsorbed onto microtiter well plastic, and conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to fibulin-1 was also observed in surface plasmon resonance assays, and a dissociation constant (Kd) of 2.9 +/- 1.6 microM was derived. In addition, fluorescence anisotropy experiments demonstrated that the interaction was also able to occur in fluid phase, which suggests that complexes of fibulin-1 and Fg could exist in the blood. To localize the portion of Fg that is responsible for interacting with fibulin-1, proteolytic fragments of Fg were evaluated for their ability to promote fibulin-1 binding. Fragments containing the carboxyl-terminal region of the Bbeta chain (residues 216-468) were able to bind to fibulin-1. In addition, it was found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed fibrin-containing thrombi associated with human atherectomy specimens. The interaction between fibulin-1 and Fg highlights potential new roles for fibulin-1 in hemostasis as well as thrombosis.

Binding Sites↗

PubMed1995

Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a cationic cradle.

The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding consensus sequence, Arg6-Arg7-Ala8-Arg9 (residues 1697-1700 in plasma fibronectin). Barkalow and Schwarzbauer (Barkalow, F.J., and Schwarzbauer, J.E. (1991) J. Biol. Chem. 266, 7812-7818) showed that mutation of Arg6-Arg7 in domain III-13 of recombinant truncated fibronectins abolished their ability to bind heparin-Sepharose. However, synthetic peptides containing this sequence have negligible affinity for heparin (Ingham, K.C., Brew, S.A., Migliorini, M. M., and Busby, T.F. (1993) Biochemistry 32, 12548-12553). We generated a three-dimensional model of fibronectin type III-13 based on the structure of a homologous domain from tenascin. The model places Arg23, Lys25, and Arg54 parallel to and in close proximity to the Arg6-Arg7-Ala8-Arg9 motif, suggesting that these residues may also contribute to the heparin-binding site. Domain III-13 and six single-site mutants containing Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli. All of the purified mutant domains melted reversibly with a Tm near that of the wild type indicating that they were correctly folded. When fluorescein-labeled heparin was titrated at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a Kd of 5-7 microM, close to that of the natural domain obtained by proteolysis of fibronectin. The R54S mutant bound 3-fold weaker and the remaining mutants bound at least 10-fold weaker than wild type. The results point out that the Arg6-Arg7-Ala8-Arg9 consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in the context of a folded domain. Thus, the heparin-binding site in fibronectin is more complex than previously realized. It is formed by a cluster of 6 positively charged residues that are remote in the sequence but brought together on one side of domain III-13 to form a "cationic cradle" into which the anionic heparin molecule could fit.

Amino Acid Sequence↗

PubMed1995

LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation.

The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.

Amyloid beta-Protein Precursor↗

PubMed1995

Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin.

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.

Animals↗

PubMed1995

The cellular internalization and degradation of hepatic lipase is mediated by low density lipoprotein receptor-related protein and requires cell surface proteoglycans.

Hepatic lipase (HL) and lipoprotein lipase (LpL) are structurally related lipolytic enzymes that have distinct functions in lipoprotein catabolism. In addition to its lipolytic activity, LpL binds to very low density lipoproteins and promotes their interaction with the low density lipoprotein receptor-related protein (LRP) (Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet M. W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). In vitro binding assays revealed that HL also binds to purified LRP with a KD of 52 nM. Its binding to LRP is inhibited by the 39-kDa receptor-associated protein (RAP), a known LRP antagonist, and by heparin. 125I-Labeled HL is rapidly internalized and degraded by HepG2 cell lines, and approximately 70% of the cellular internalization and degradation is blocked by either exogenously added RAP or anti-LRP IgG. Mouse fibroblasts that lack LRP display a greatly diminished capacity to internalize and degrade HL when compared to control fibroblasts. These data indicate that LRP-mediated cellular uptake of HL accounts for a substantial portion of the internalization of this molecule. Proteoglycans have been shown to participate in the clearance of LpL, and consequently a role for proteoglycans in HL clearance pathway was also investigated. Chinese hamster ovary cell lines that are deficient in proteoglycan biosynthesis were unable to internalize or degrade 125I-HL despite the fact that these cells express LRP. Thus, the initial binding of HL to cell surface proteoglycans is an obligatory step for the delivery of the enzyme to LRP for endocytosis. A small, but significant, amount of 125I-HL was internalized in LRP deficient cells indicating that an LRP-independent pathway for HL internalization does exist. This pathway could involve cell surface proteoglycans, the LDL receptor, or some other unidentified surface protein.

Animals↗

PubMed1995

Identification of a novel form of the alpha 3 integrin subunit: covalent association with transferrin receptor.

The alpha 3 beta 1 integrin is a cell-surface receptor for laminin, entactin, collagen, fibronectin and epiligrin. On some prostatic-carcinoma cell lines that express the alpha 3 beta 1 heterodimer we have identified a novel form of the alpha 3 subunit. Whereas the prototypic alpha 3 subunit has a molecular mass of approximately 155 kDa, we have isolated a approximately 225 kDa protein (p225) which is recognized by monoclonal antibodies to the alpha 3 subunit. Protein sequence analysis revealed that p225 consists of two polypeptides, namely integrin alpha 3 heavy chain (approximately 130 kDa) disulphide-bonded to a monomer of the transferrin receptor (approximately 95 kDa) instead of the typical alpha 3 light chain (approximately 25 kDa). The p225 seems to be directly associated with beta 1 subunit, since it was immunoprecipitable with anti-(beta 1 subunit) antibodies. The association of transferrin receptor and integrin alpha 3 was apparently not the result of spurious disulphide-bond formation occurring during the protein purification, as iodoacetamide and GSH did not block the formation of the complex. The transferrin receptor is normally a homodimer that is involved in the internalization of iron-bound transferrin into cells and can be expressed at relatively high levels in the cell lines which we have studied. The p225 is not found on all cell types examined to date and therefore it may represent a unique complex between the integrin alpha 3 subunit and the transferrin receptor, a covalent association which may play a role in the adherence and/or proliferation of some types of tumour cells.

Amino Acid Sequence↗

PubMed1995

A quantitative analysis of the incorporation of fibulin-1 into extracellular matrix indicates that fibronectin assembly is required.

Fibulin-1 is an extracellular matrix glycoprotein found in both loose and dense connective tissues, elastic fibers and some basement membranes. Cultured cells such as fibroblasts assemble endogenously synthesized or exogenously added fibulin-1 into matrix fibrils that also contain fibronectin. Since we have previously shown that fibulin-1 binds to fibronectin (Balbona, K., Tran, H., Godyna, S., Ingham, K. C., Strickland, D. K. and Argraves, W. S. J. Biol. Chem. 267: 20120-20125, 1992), we sought to investigate fibulin-1 incorporation into fibroblast extracellular matrix with an emphasis on evaluating the potential role of fibronectin in the process. In this study, we have used quantitative assays to measure the binding of 125I-fibulin to monolayers of cultured fibroblasts. Our results show that the kinetics of fibulin-1 incorporation into the cell layer and its partitioning into detergent-soluble and -insoluble fractions were similar to those of fibronectin. It was found that antibodies to fibronectin or to the fibulin-1-binding domain of fibronectin-inhibited fibulin-1 incorporation. Cell lines that fail to assemble fibronectin into the matrix, such as HT1080 or PFHR-9, do not incorporate fibulin-1 into their cell layers. However, when HT1080 cells were induced to assemble fibronectin by treatment with dexamethasone, they subsequently acquired the ability to incorporate fibulin-1. Moreover, treatment of cultured fibroblasts with antibodies that inhibit fibronectin assembly significantly inhibit fibulin-1 incorporation into the matrix. When increased amounts of fibronectin were incorporated into cells layers by incubating the cells for varying lengths of time with exogenous fibronectin, a corresponding increase in fibulin-1 incorporation was also observed. Taken together, the data indicate that the incorporation of fibulin-1 requires fibronectin assembly and suggests a dependence on the amount of fibronectin in a matrix. These results highlight the potential of fibronectin to control the deposition of fibulin-1 into those extracellular matrices where both proteins coincide and may have implications in the formation of fibulin-1-containing matrix structures such as basement membranes or elastic fibers.

Antibodies↗

PubMed1995

Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1.

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre-treatment of the cells with either heparitinase, chondroitinase or beta-D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.

Binding, Competitive↗

PubMed1995

LDL receptor-related protein: a multiligand receptor for lipoprotein and proteinase catabolism.

The accumulation of excessive cholesterol-rich lipoproteins within vascular cells, the proliferation of vascular cells, and fibrin deposition are hallmark features of atherosclerosis. Evidence accumulated over the past few years supports the hypothesis that one member of the LDL receptor family, the low density lipoprotein receptor-related protein (LRP), affects the dynamics of each of these processes. LRP is expressed in several vascular cell types, including smooth muscle cells, and in macrophages, and is also expressed in these cells in atherosclerotic lesions. This receptor is a large endocytotic receptor that mediates the catabolism of a number of molecules known to be important in vascular biology, including apolipoprotein E- and lipoprotein lipase-enriched lipoproteins, thrombospondin, and plasminogen activators. The capacity of LRP to mediate lipoprotein catabolism may be a factor in the development of the lesion by contributing to the formation of foam cells. LRP has recently been shown to mediate the catabolism of thrombospondin, a molecule that has potent biological effects on cells of the vasculature. The regulation of its extracellular accumulation by LRP might modulate the dynamic processes of tissue remodeling associated with the response to vascular injury. In addition, LRP regulates the expression of plasmin activity by directly binding and mediating the cellular internalization of urokinase- and tissue-type plasminogen activators. The cellular removal of these two enzymes decreases the local profibrinolytic potential, possibly leading to a thrombotic state at lesion sites.

Endocytosis↗

PubMed1995

Localization of the human gene for fibulin-1 (FBLN1) to chromosome band 22q13.3.

Using fibulin-1 cDNA probes, we performed fluorescence in situ hybridization to map the human chromosomal location of the gene encoding the extracellular matrix and blood glycoprotein, fibulin-1 (FBLN1). The gene for fibulin-1 was mapped to a single site on the long arm of human chromosome 22 (22q13.3). The assignment of the chromosomal map position for the fibulin-1 gene will aid in the evaluation of its potential roles in human connective tissue and blood diseases.

Calcium-Binding Proteins↗