Hyaluronate-cell interactions during differentiation of chick embryo limb mesoderm

The mechanism of interaction of hyaluronate with the surface of cells from embryonic chick limbs was studied using cell cultures of mesoderm from various developmental stages. The mode of interaction of hyaluronate with the cell surface changed at the onset of mesodermal cell condensation prior to differentiation of cartilage and muscle. At this time hyaluronate binding sites appeared on the cells and continued to be present on differentiated chondrocytes but not on myotubes. Direct measurement of hyaluronate binding was made using stage 24 mesodermal cells and membranes isolated from cells derived from various limb stages. The stage 24 cells and membranes from stage 22, 24, and 26 cells exhibited hyaluronate binding, but not membranes from stage 19 mesoderm cultures. At stage 38, membranes from chondrocyte cultures exhibited the highest hyaluronate binding, and membranes from myoblasts and fibroblasts intermediate binding, whereas membranes from myotube-enriched cultures lacked binding activity. No significant competition of hyaluronate binding by chondroitin sulfate was observed. Occupied hyaluronate binding sites were measured by the displacement of radiolabeled cell surface hyaluronate with exogenous, unlabeled hyaluronate. Very little hyaluronate was displaced from mesodermal cells derived from the youngest embryos, namely, stage 19 or stage 20-21. However, greater than 50% of cell surface hyaluronate was displaced from stage 22 and 24 mesodermal cells. The addition of exogenous hyaluronate to stage 26 mesoderm, the stage of onset of cartilage differentiation, and to stage 38 chondrocytes resulted in displacement of large proportions of both hyaluronate and chondroitin sulfate. Addition of exogenous chondroitin sulfate did not cause displacement of significant amounts of cell surface hyaluronate or chondroitin sulfate. These results indicate the presence and developmental modulation of specific binding sites for hyaluronate on limb cells during their differentiation.     

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.     

The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins [published erratum appears in J Cell Biol 1991 Feb;112(3):following 513]

The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells. CD44 has also been implicated in lymphocyte binding to high endothelial venules during lymphocyte homing. Interestingly, the monoclonal antibody Hermes- 3, which blocks lymphocyte binding to the high endothelial venules of mucosal lymphoid tissue, had no effect on the binding of labeled hyaluronate. Furthermore, the binding of lymphocytes to high endothelial cells of lymph nodes and mucosal lymphoid tissue was not significantly affected by treatment with agents that block the binding of hyaluronate (hyaluronidase, excess hyaluronate and specific antibodies). Thus, CD44 appears to have at least two distinct functional domains, one for binding hyaluronate and another involved in interactions with mucosal high endothelial venules.     

Hyaluronate binding properties of versican.

We have previously cloned a large chondroitin sulfate proteoglycan (versican) from human fibroblasts. The primary sequence shows that the N terminus contains sequence homology with known hyaluronate-binding molecule, suggesting that versican can bind hyaluronate. To test this hypothesis we have reconstructed a full-length versican cDNA and a versican cDNA fragment encoding the N terminus and have transfected Chinese hamster ovary cells and mouse 3T3 fibroblasts, respectively, with these constructs. The transfected Chinese hamster ovary cells make a proteoglycan shown to be versican by enzymatic and immunologic analysis. No corresponding proteoglycan was seen in the control cells. Using hyaluronate affinity chromatography, we show that recombinant versican specifically binds hyaluronate and does not bind to heparin or chondroitin sulfate. The transfected fibroblasts make a 78-kDa truncated form of versican that also binds hyaluronate and does not bind the related polysaccharides, showing that the hyaluronate binding activity resides at the N terminus of versican. The binding of versican to hyaluronate is substrate-concentration dependent and time dependent and can be competed with unlabeled versican. The dissociation constant for versican binding to hyaluronate was determined to be 4 x 10(-9) M.     

Binding of hyaluronate to the surface of cultured cells

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.     

Membrane-associated hyaluronate-binding activity of chondrosarcoma chondrocytes

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyces hyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H-acetate, to exogenous hyaluronate or to Streptomyces hyaluronidase resulted in the release of 36-38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind 3H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd = approximately 10(-10) M), and destroyed by treating the membranes with trypsin. The 3H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyaluronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000-66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to monoclonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreactive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.     

Characterization and identification of the hyaluronate binding site from membranes of SV-3T3 cells

Previous research has shown that binding sites for hyaluronate are present on the surfaces of a number of different cell types. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and dissolved in a solution of sodium deoxycholate. Hyaluronate binding activity was detected by mixing the sodium deoxycholate extract with [3H]hyaluronate and then adding an equal volume of saturated (NH4)2SO4, which precipitated the binding protein and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. Following partial purification by hydroxylapatite chromatography, the binding site was examined by molecular sieve chromatography and by rate-zonal centrifugation, which revealed that it has a Stokes radius of 6.5 nm and a sedimentation coefficient of 4.8 S. From these values, it was possible to calculate that the sodium deoxycholate-solubilized binding site has a frictional coefficient of 1.87 and a molecular weight of 132,000. Since this latter value applies to the complex of both detergent and protein, the binding protein by itself must have a molecular weight lower than 132,000. To determine the molecular weight of the hyaluronate binding site itself, the protein was purified by the sequential application of hydroxylapatite chromatography, molecular sieve chromatography, rate-zonal centrifugation, and finally lectin-affinity chromatography on concanavalin A-agarose. Analysis of the purified material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85,000 Mr protein which has been identified as the binding site. This protein was also detected on nitrocellulose blots which had been specifically stained for concanavalin A binding material, suggesting that the binding site is a glycoprotein.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44- hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44- mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N- linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.     

Localization of hyaluronate in primary glial cell cultures derived from newborn rat brain

We have devised a technique that enables one to localize hyaluronate in cultured cells. Cells were probed with the glial hyaluronate binding protein (GHAP) which was itself then visualized by conventional indirect immunofluorescence. The hyaluronate binding properties of this protein have been established. This technique was applied to the study of hyaluronate synthesis in glial cells. These cells do not themselves produce GHAP. O-2A progenitor cells were obtained from the cerebral hemispheres of newborn rats. These cells are bipotential in that they are able to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium. In cultures of O-2A progenitor cells maintained in the absence of serum, in which large numbers of oligodendrocytes appeared, very little hyaluronate was produced. The galC+ cells were invariably hyaluronate negative. Cultures of the same cells, maintained in the presence of 10% FCS, contained large numbers of hyaluronate producing cells. The hyaluronate producing cells were typically small, process-bearing, and GFAP+. Some, but not all, were A2B5+ and could, therefore, be identified as type 2 (GFAP+, A2B5+) astrocytes. Type 1 (GFAP+, A2B5-) astrocytes were also active in the synthesis of hyaluronate, to the extent that they were able to coat their substrate with hyaluronate. Among cells of the O-2A lineage, then, hyaluronate production would appear to be restricted to astrocytes. This may have some bearing on the origin of hyaluronate in the extracellular matrix of CNS white matter.     

Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition

Purified human tumor necrosis factor (TNF) was iodinated to high specific activity with good retention of its biological activity, as determined by the cytotoxic titer on murine L929 cells. The binding of 125I-TNF to L929 and human HeLa S2 cells grown in monolayer was initially measured, but high levels of nonspecific binding were observed. Specific binding to high affinity receptors of HeLa S2 cells grown in suspension culture was demonstrated by competitive displacement experiments and analysis of the binding data with the LIGAND program. A KD of 2 X 10(-10) M and 6000 receptors/cell were calculated in this way. These observations provide the first direct evidence for a cellular receptor for TNF. The cell-bound 125I-TNF was internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and subsequently degraded to acid-soluble products. Three lines of human lymphoblastoid cells were examined for sensitivity to the cytostatic effect of TNF and for the presence of high affinity receptors. Daudi and Raji cells were insensitive to TNF and showed very few specific binding sites when incubated with 125I-TNF. Jurkat cells were growth-inhibited by TNF and showed a significantly greater number of specific binding sites than the other lymphoblastoid cells. These findings suggest that the sensitivity of some cell lines to the biological effects of TNF may be correlated with the presence of a relatively high number of receptors for this factor.     

Mononuclear cell-mediated modulation of synovial cell metabolism. II. Increased hyaluronic acid synthesis by a monocyte cell factor (MCF)

Treatment of cultured human synovial cells with a mononuclear cell factor (MCF) enhanced their ability to synthesize glycosaminoglycans (GAG), but GAG repartition between extracellular, pericellular and intracellular compartments was found to be the same as in control. Hyaluronic acid (HA) production, which represents 80-90% of all secreted GAG, was stimulated 2 1/2-3-fold, but the HA molecular weight was not modified. The MCF increased the hyaluronate synthetase activity of synovial cells in similar proportions. Actinomycin D inhibited the increase in hyaluronate synthetase activity produced by MCF, indicating that this increase involves new synthesis of mRNA. Stimulation of both HA synthesis and hyaluronate synthetase activity by MCF was suppressed by 10(-4)-10(-5) M indomethacin (an inhibitor of cyclo-oxygenase), suggesting that MCF effect is prostaglandin-dependent.     

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (Mr,e) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with [35S]methionine label. The three protein species isolated by hyaluronate affinity chromatography (Mr,e 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of 3H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The apparent dissociation constant of HABP for hyaluronate was approximately 10(-8) M, and kinetic analyses showed these binding interactions were complex and of a positive cooperative nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin and heparan sulfate binding sites on B-16 melanoma cells

We have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast-deposited matrix component, possibly heparan sulfate-proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B-16 melanoma cells have been demonstrated. Binding of 3H-heparin and 35S-heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H-heparin yielded a Kd of 2-5 x 10(-8) M and a Bmax of 0.5 x 10(7) heparin molecules bound per cell. Binding of 35S-heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S-heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H-heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H-heparin to transblots of the detergent extract of the B-16 cell membranes indicated that at least part of the binding activity is a 14,000-dalton protein.     

Subcellular localization of hyaluronate synthetase in oligodendroglioma cells

In order to provide some insight into the mechanism of hyaluronate synthesis, the subcellular localization of the synthetase system for hyaluronate was determined in eukaryotic cells. The mouse oligodendroglioma cell line G26-24, which produces copious amounts of hyaluronate in culture, was chosen as a system for these studies. Protease treatment and homogenization of cells followed by hyaluronate synthetase assay suggested that nucleotide-binding sites and trypsin-sensitive synthetase sites were not exposed at the outer membrane surface. Protease treatment following homogenization did result in decreased activity. Membrane fragments, prepared by gentle homogenization in iso- and hypotonic buffers, were subjected to differential centrifugation followed by several continuous and discontinuous sucrose equilibrium and velocity gradient systems. Hyaluronate synthetase activity co-fractionated with a plasma membrane marker in all systems, including those in which Golgi markers were separable. Treatment of intact cells in culture with several hyaluronidases resulted in a marked stimulation of cell-free synthetase activity. The stimulated activity was also found exclusively in plasma membrane-enriched fractions.     

Vitamin C inhibits the enzymatic activity of Streptococcus pneumoniae hyaluronate lyase

Enzyme activity measurement showed that L-ascorbic acid (vitamin C (Vc)) competitively inhibits the hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. The complex crystal structure of this enzyme with Vc was determined at 2.0 A resolution. One Vc molecule was found to bind to the active site of the enzyme. The Vc carboxyl group provides the negative charges that lead the molecule into the highly positively charged cleft of the enzyme. The Vc ring system forms hydrophobic interactions with the side chain of Trp-292, which is one of the aromatic patch residues of this enzyme responsible for the selection of the cleavage sites on the substrate chain. The binding of Vc inhibits the substrate binding at hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The high concentration of Vc in human tissues probably provides a low level of natural resistance to the pneumococcal invasion. This is the first time that Vc the direct inhibition on the bacterial "spreading factor" was reported, and Vc is also the first chemical that has been shown experimentally to have an inhibitory effect on bacterial hyaluronate lyase. These studies also highlight the possible structural requirement for the design of a stronger inhibitor of bacterial hyaluronate lyase.     

Binding of hyaluronate and chondroitin sulphate to liver endothelial cells.

Hyaluronate is taken up and metabolized in liver endothelial cells by means of a receptor. To characterize the interaction with the receptor, two preparations of 3H-labelled hyaluronate, of Mr 4 X 10(5) and 6.4 X 10(6), and a series of hyaluronate oligosaccharides were bound to cultured liver endothelial cells at 7 degrees C. The dissociation constant varied between 4.6 X 10(-6) M for an octasaccharide and 9 X 10(-12) M for the largest polymer. The Mr-dependence for the series of oligosaccharides was explained by the increased probability of binding due to the repetitive sequence along the chain. The high affinity of high-Mr hyaluronate for the receptor could also be mainly ascribed to this effect, which rules out any major contribution of co-operative multiple-site attachment to the cell surface. Each liver endothelial cell can bind 10(5) oligosaccharides, about 10(4) molecules with Mr 4 X 10(5) and about 10(3) molecules with Mr 6.4 X 10(6). This is explained by mutual exclusion of large molecules from the cell surface. Chondroitin sulphate is also bound to liver endothelial cells. Inhibition studies showed that it binds to the same receptor as hyaluronate and with an affinity that is about 3-fold higher than that of hyaluronate of the same degree of polymerization.     

Characterization of plasminogen binding to human capillary and arterial endothelial cells.

Phenotypic diversity of endothelial cells that line the various vascular spaces has been well established. However, it is not known if biochemical differences also exist, particularly in the numbers of receptors for plasma proteins. Equilibrium binding techniques were used to assess potential differences in the binding of 125I-labelled plasminogen to cultured human umbilical arterial endothelial cells and capillary endothelium, as compared with umbilical venous cells. The kinetic behaviour of plasminogen binding to all three types of cells was similar, with optimal binding occurring between 20 and 30 min of incubation. Binding of plasminogen to arterial, capillary, and venous cells was concentration dependent and reversible upon addition to excess unlabelled plasminogen. Scatchard analyses showed that artery, capillary, and venous endothelial cells all possess low affinity sites for plasminogen with Kd values of 0.30 +/- 0.07, 0.40 +/- 0.06, and 0.40 +/- 0.08 microM, respectively. Vein cells also possess an additional higher affinity binding site with a Kd of 0.07 +/- 0.01 microM, exhibiting a 6-fold greater affinity for plasminogen than the lower affinity sites on capillary and arterial endothelial cells. Assuming a stoichiometry of 1:1 for binding, the data indicate that arterial and capillary endothelial cells contain approximately 4.2 (+/- 0.9) x 10(6) and 4.1 (+/- 0.6) x 10(6) plasminogen receptors per cell. Venous cells contain both low and high density binding sites with 6.2 (+/- 0.8) x 10(6) and 12.4 (+/- 2.4) x 10(6) sites per endothelial cell. The presence of a higher affinity site on vein cells, but not on artery or capillary cells, may signal functional differences relating to fibrinolytic activity on the surface of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.