Characterization of a membrane receptor for transforming growth factor-beta in normal rat kidney fibroblasts

A procedure has been developed for the iodination of human transforming growth factor-beta (TGF-beta) with full retention of biological activity. Using the iodinated peptide, saturable receptors have been found for TGF-beta on normal rat kidney fibroblasts, a cell line that will grow in soft agar in the presence of TGFs but not in their absence. Scatchard analysis of the binding data showed a high affinity binding site (dissociation constant equal to 25 to 30 pM with approximately 17,000 receptors per cell). The receptor was specific for TGF-beta with epidermal growth factor, insulin, insulin-like growth factors I and II, platelet-derived growth factor, and TGF-alpha being unable to compete for the binding of 125I-TGF-beta to the receptor. The binding of TGF-beta was a time- and temperature-dependent process. At 37 degrees C, maximal binding was attained within 45 to 60 min after addition of 125I-TGF-beta followed by a rapid decline in cell-associated radioactivity due to degradation of the 125I-TGF-beta. As demonstrated using ammonium acetate, a compound known to inhibit lysosomal enzymes, this degradation was most likely due to the action of proteolytic enzymes found in the lysosome. At 0 degrees C, binding reached a plateau within 2 to 3 h and maintained this level with no apparent drop during the 4-h incubation period. The receptor could be down-regulated by TGF-beta, but not by epidermal growth factor, to approximately 50% of the level initially observed.     

Vascular smooth muscle cells express distinct transforming growth factor-beta receptor phenotypes as a function of cell density in culture

Transforming growth factor-beta (TGF-beta) is a bifunctional, density-dependent regulator of vascular smooth muscle cell (SMC) proliferation in vitro (at sparse densities SMC are growth-inhibited by the peptide, whereas at confluent densities TGF-beta potentiates their growth). We have used affinity labeling and ligand binding techniques to characterize cell surface receptors for TGF-beta under sparse and confluent culture conditions. Confluent SMC, whose growth are promoted by TGF-beta, exhibited a single class of high affinity TGF-beta binding sites (Kd = 6 pM, 3,000 sites/cell). In contrast, sparse SMC (whose growth are inhibited by TGF-beta) expressed two distinct classes of high affinity binding sites with binding constants of 6 pM (3,000 sites/cell) and 88 pM (11,000 sites/cell). By affinity labeling using 125I-TGF-beta and disuccinimidyl suberate cross-linking, confluent cells were found to express a major Mr = 280,000 TGF-beta receptor as well as trace amounts of low molecular weight (Mr = 85,000 and 65,000) receptor subtypes. All three of these receptors were determined, by ligand competition, to show similar affinity for TGF-beta. The predominant receptor subtypes expressed by sparse SMC exhibited apparent Mr = 75,000 and 65,000. In ligand competition experiments, the Mr = 75,000 receptor subtype (never present in confluent cultures) exhibited lower relative affinity for TGF-beta than did the Mr = 65,000 form. The ability of TGF-beta to inhibit SMC proliferation, therefore, correlates with the expression of a unique TGF-beta-binding protein on the SMC surface. The data suggest that TGF-beta may exert opposite biological effects on the same cell type via an interaction with distinct, selectively expressed receptor subtypes.     

Preparation and binding of radioactively labeled porcine transforming growth factor type beta

This report describes the labeling of porcine transforming growth factor type beta (TGF-beta) with 125-iodine. Its binding to NRK cells and three other cell lines has been examined. The data indicate that NRK cells exhibit approximately 10,000 receptors for porcine TGF-beta per cell, with an apparent dissociation constant of 45 pM. The binding of porcine 125I-TGF-beta can be blocked by porcine, murine and human TGF-beta but not by several well characterized growth factors. In all respects examined, the binding observed with porcine 125I-TGF-beta appears to be the same as that observed with human TGF-beta. The findings reported here argue that porcine 125I-TGF-beta can be used to quantitate TGF-beta receptors on a wide range of mammalian cells.     

Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.

Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta 1 and TGF-beta 2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta 1 and TGF-beta 2. The Kd values obtained from Scatchard analyses were approximately 65 pM for 125I-TGF-beta 1 and approximately 40 pM for 125I-TGF-beta 2, with 70,000 and 85,000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta 1 and TGF-beta 2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 125I-TGF-beta 1 and 125I-TGF-beta 2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta 1 than for -beta 2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta 2 than for -beta 1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta 2 than for -beta 1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.     

Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.     

A surface component on GH3 pituitary cells that recognizes transforming growth factor-beta, activin, and inhibin

We have examined the ability of various forms of activin and inhibin, which are structurally related to transforming growth factor-beta (TGF-beta), to interact with various types of cell surface TGF-beta binding sites. Activin AB, inhibin A, and inhibin B were unable to compete with 125I-TGF-beta 1 for binding to the TGF-beta receptor types I, II, or III that coexist in human skin fibroblasts, rat liver epithelial cells, and mink lung epithelial cells. In contrast, activins and inhibins effectively competed for TGF-beta 1 binding to GH3 rat pituitary tumor cells. Binding of TGF-beta 1 to GH3 cells was mediated by about 2700 sites/cell with a Kd = 90 pM. Affinity labeling of these GH3 binding sites by cross-linking to 125I-TGF-beta 1 yielded 70-74-kDa labeled complexes distinct from previously identified TGF-beta binding components. Labeling of these 70-74-kDa components with 125I-TGF-beta 1 was inhibited by TGF-beta 1, TGF-beta 2, activin AB, and inhibin B at concentrations in the high picomolar to low nanomolar range, but it was not significantly affected by other polypeptide hormones and growth factors tested. The 70-74-kDa labeled GH3 components represent a novel type of cell surface TGF-beta binding protein that is unique in its ability to recognize various other members of the TGF-beta family of bioactive polypeptides.     

Modulation of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors.

A single class of high-affinity receptors for EGF were detected on mouse embryonic palatal mesenchyme (MEPM) cells cultured in vitro. The degree of confluence of the cultured cells did not affect the number or affinity of the binding sites. Culture of MEPM cells in the presence of bFGF, IGF-II or TGF-beta 1 induced changes in 125I-EGF binding. TGF-beta 1 caused a marked reduction in binding to 40% of control levels. This reduction was achieved after 2 h and persisted for 24 h after addition of the growth factor. IGF-II induced a similar reduction but this effect was transitory; after a 12 h pretreatment with IGF-II, binding was restored to control levels. The effects of bFGF were biphasic. Initially, a short pre-treatment period (3-5 h) with bFGF caused a small reduction in 125I-EGF binding; longer periods of pre-incubation (24 h) resulted in a large increase in receptor number. Pre-incubation in medium containing both bFGF and TGF-beta 1 resulted in a decrease in EGF binding. Thus, TGF-beta 1 negated the large increase in receptor number induced by bFGF alone. Changes in receptor number were usually, but not always, directly related to changes in the biological activity of EGF, as assessed by a thymidine incorporation assay. This study highlights the possible interactive role of growth factors known to be present in the developing palate.     

A transforming growth factor beta (TGF-beta) receptor from human placenta exhibits a greater affinity for TGF-beta 2 than for TGF-beta 1

Affinity-labeling techniques have been used to identify three types of high-affinity receptors for transforming growth factor beta (TGF-beta) on the surface of many cells in culture. Here we demonstrate that membrane preparations from tissue sources may also be used as an alternative system for studying the binding properties of TGF-beta receptors. Using a chemical cross-linking technique with 125I-TGF-beta 1 and 125I-TGF-beta 2 and bis(sulfosuccinimidyl)suberate (BS3), we have identified and characterized two high-affinity binding components in membrane preparations derived from human term placenta. The larger species, which migrates as a diffuse band of molecular mass 250-350 kDa on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, is characteristic of the TGF-beta receptor type III, a proteoglycan containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. The smaller species of molecular mass 140 kDa was identified as the core glycoprotein of this type III receptor by using the techniques of enzymatic deglycosylation and peptide mapping. Competition experiments, using 125I-TGF-beta 1 or 125I-TGF-beta 2 and varying amounts of competing unlabeled TGF-beta 1 or TGF-beta 2, revealed that both the placental type III proteoglycan and its core glycoprotein belong to a novel class of type III receptors that exhibit a greater affinity for TGF-beta 2 than for TGF-beta 1. This preferential binding of TGF-beta 2 to placental type III receptors suggests differential roles for TGF-beta 2 and TGF-beta 1 in placental function.     

Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells.

Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.     

Decrease in transforming growth factor beta 1 binding during differentiation of rat adipocyte precursors in primary culture

Transforming growth factor beta 1 (TGF-beta 1) binding and action were investigated during differentiation of adipocyte precursors freshly isolated from rat inguinal fat-pad cultivated in defined medium. The data presented in this paper indicate that TGF-beta 1 inhibits differentiation of adipocyte precursors with a 50% effective dose of 9 pM. Time course experiments demonstrate that TGF-beta 1 is active only when it is added to the cells while they are still undifferentiated. If added after the cells have started to differentiate, TGF-beta 1 is less active or becomes inactive. 125I-TGF-beta 1 binding studies on adipocyte precursors before and after differentiation indicate a 10-fold decrease in the number of TGF-beta 1 binding sites after the cells have differentiated. Blocking of the differentiation process by treating the cells with fetal bovine serum or with prostaglandin F2 alpha prevented the decrease in the number of TGF-beta 1 receptors, thereby demonstrating that this change in binding was specifically linked to the differentiation process. Experiments cross-linking 125I-TGF-beta 1 to adipocyte precursors showed that 125I-TGF-beta 1 is specifically cross-linked to two bands with molecular weights of 92,000 and 70,000. After differentiation, a decrease in the intensity of the cross-linked bands was observed. These results demonstrate that loss of cell surface TGF-beta 1 binding sites follows differentiation of adipocyte precursors.     

Bifunctional effects of transforming growth factor-beta (TGF-beta) on endothelial cell growth correlate with phenotypes of TGF-beta binding sites.

Transforming growth factor-beta (TGF-beta) is a bifunctional, dose-dependent regulator of endothelial cell proliferation induced in vitro by heparin-binding growth factor 1 (HBGF-1, acidic FGF). Here we have examined the relationship between endothelial cell growth and the expression of cell surface binding sites for TGF-beta and HBGF-1. Fetal bovine heart endothelial cell (FBHEC) growth was stimulated by low concentrations of TGF-beta and inhibited by high concentrations of TGF-beta while expressing two distinct classes of TGF-beta binding sites with binding constants of 24 pM (6300 sites/cell) and 900 pM (12,000 sites/cell). In contrast, human umbilical vein endothelial cells (HUVEC), whose growth was slightly promoted by TGF-beta, exhibited a single class of high-affinity TGF-beta binding sites (Kd = 45 pM, 4500 sites/cell). Affinity crosslinking using [125I]TGF-beta showed that FBHEC expressed two distinct low molecular weight TGF-beta binding sites (Mr 85,000 and 58,000), while HUVEC expressed a single type of low molecular weight TGF-beta binding site (Mr 85,000). As detected by binding of [125I]HBGF-1, preincubation of FBHEC with high concentrations of TGF-beta transmodulated the expression of high-affinity HBGF-1 receptors. In contrast, no transmodulation of HBGF-1 receptors occurred in FBHEC during preincubation with low concentrations of TGF-beta. Furthermore, preincubation of HUVEC with TGF-beta did not transmodulate the expression of HBGF-1 receptors. The data suggest that the ability of TGF-beta to stimulate or inhibit endothelial cell proliferation in a dose-dependent manner correlated with the expression of specific TGF-beta binding site subtypes and involved the transmodulation of HBGF-1 receptors.     

Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells.

Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin.     

A binding assay for the solubilized receptors of type beta transforming growth factor: adsorption and removal of free ligand by dextran-coated charcoal

A binding assay was developed for the measurement of solubilized receptors for transforming growth factor type beta (TGF-beta). Solubilized receptors were incubated with 125I-TGF-beta, then the unbound ligand was removed by adsorption to dextran-coated charcoal. The binding of TGF-beta to solubilized receptors was saturable and specific, and increased in a linear manner with respect to the amount of membrane protein present. Crosslinking of radioactive complexes after adsorptive removal of unbound TGF-beta yielded complexes similar to affinity-labeled TGF-beta receptors from whole cells. Treatment of a 20% charcoal suspension with 0.2-0.4% dextran was optimal for the protection of receptors from adsorption to charcoal while allowing free TGF-beta to be removed; Mr approximately 250,000 dextran was most effective. This method can assay receptors from purified membranes and crude extracts of cells and tissues, and was used to demonstrate that TGF-beta receptors are glycosylated and retain a high affinity (Kd approximately 530 pM) for ligand after solubilization.     

Purification of a new type high molecular weight receptor (type V receptor) of transforming growth factor beta (TGF-beta) from bovine liver. Identification of the type V TGF-beta receptor in cultured cells.

A 400-kDa transforming growth factor beta (TGF-beta) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B affinity chromatography, DEAE-cellulose anion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. This procedure yielded approximately 20 micrograms of the receptor from 1 kg of bovine liver. During purification, the 400-kDa TGF-beta receptor was detected by a cross-linking assay in which the TGF-beta receptor-125I-TGF-beta complex was cross-linked by disuccinimidyl suberate, a bifunctional reagent, and analyzed by 5.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. This novel 400-kDa TGF-beta receptor was also identified on cultured cells including cells reported to lack the type III receptor. The 400-kDa TGF-beta receptor, a nonproteoglycan glycoprotein, appears to be distinct from TGF-beta receptors (types I, II, III, and IV) previously identified on cultured cells and is designated as the type V receptor. The 400-kDa TGF-beta receptor as well as type I, II, and III receptors underwent internalization upon 125I-TGF-beta binding in mink lung epithelial cells.     

Structure and properties of the cellular receptor for transforming growth factor type beta

Swiss 3T3 cells respond to picomolar concentrations of type beta transforming growth factor (TGF-beta) with a dose-dependent increase in the formation of colonies in soft agar, a decrease in the growth of cells in monolayer culture, and changes in morphology. This indicates that these cells have functional TGF-beta receptors able to mediate a biological response. Binding analysis revealed a single class of TGF-beta binding sites (80 000 per cell) with a Kd approximately 50 pM. Receptors were affinity-labeled by covalent attachment to 125I-TGF-beta with bis(sulfosuccinimidyl) suberate (BS3). The complexes formed were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 100 mM dithiothreitol and migrated as Mr approximately 180 000 complexes in 3-10% linear gradient gels. The apparent size of these complexes was larger in gels with a higher percentage of acrylamide. The labeling of the 125I-TGF-beta-receptor complexes was inhibited by the presence of excess unlabeled TGF-beta but was unaffected by other growth factors. These complexes could be formed by cross-linking whole cells, intact membranes, or solubilized membranes, demonstrating that the TGF-beta receptor is located on the plasma membrane and can be solubilized without destruction of its ability to bind TGF-beta. A larger Mr approximately 360 000 complex was present in 3-10% linear gradient gels without reduction or after extensive cross-linking, suggesting that the receptor consists of two subunits of similar size attached by disulfide bonds. Since BS3 is membrane-impermeable, at least a portion of both subunits is located on the outer surface of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterodimeric transforming growth factor beta. Biological properties and interaction with three types of cell surface receptors

Type beta transforming growth factors (TGF) are disulfide-linked homo- and heterodimers of two related polypeptide chains, beta 1 and beta 2. The homodimers TGF-beta 1 and TGF-beta 2 are widely distributed, but the heterodimer TGF-beta 1.2 has been found only in porcine platelets (Cheifetz, S., Weatherbee, J.A., Tsang, M.L.-S., Anderson, J.K., Mole, J.E., Lucas, R., and Massagué, J. (1987) Cell 48, 409-415). Here we characterize the receptor binding and biological properties of TGF-beta 1.2 and compare them with those of TGF-beta 1 and TGF-beta 2. Three types of cell surface receptors previously identified by affinity labeling with 125I-TGF-beta 1 are available for binding to TGF-beta 1.2. These three types of receptors are detected as 65-kDa (type I), 85-95-kDa (type II), and 250-350-kDa (type III) affinity-labeled receptor complexes on electrophoresis gels. They co-exist in many cell types, have high affinity for TGF-beta 1, and varying degrees of affinity for TGF-beta 2. Of the 11 cell lines screened in the present study none showed evidence for additional receptor types that would bind TGF-beta 2 but not TGF-beta 1. In receptor competition studies, TGF-beta 1, TGF-beta 1.2, and TGF-beta 2 competed for binding to type I and type II receptors with a relative order of potencies of 16:5:1 and 12:3:1, respectively, whereas all three forms of TGF-beta were equipotent as ligands for the type III receptors. The three forms of TGF-beta were equally potent at stimulating the biosynthesis of extracellular sulfated proteoglycan in BRL-3A rat liver epithelial cells, a response that presumably involves the type III receptor present in these cells. In contrast, the ability of the three ligands to inhibit the growth of B6SUt-A multipotential hematopoietic progenitor cells which display only type I receptors decreased in the order TGF-beta 1, TGF-beta 1.2, and TGF-beta 2 with a relative potency of 100:30:1. The results indicate that the presence of one beta 1 chain in TGF-beta 1.2 increases (with respect to TGF-beta 2) the biological potency and binding affinity toward receptor types I and II, but the presence of a second beta 1 chain in the dimer is required for full potency.     

Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.     

Further studies of the role of transforming growth factor-beta in human B cell function

This study was designed to address three specific questions in human B cells. First, to determine whether transforming growth factor-beta (TGF-beta)2 has similar biologic effects on B cell function as does TGF-beta 1. Second, to test the hypothesis that TGF-beta 1 is an autocrine growth and differentiation inhibitor. Finally, because multiple receptor species for TGF-beta have been identified on other cell types, to determine by chemical cross-linking and competitive binding studies the nature of the TGF-beta 1 R present on normal and transformed B cells. Exogenous TGF-beta 2 was found to be functionally similar to TGF-beta 1 in its inhibition of factor dependent normal B cell proliferation and Ig secretion. When an antibody, specific for the active form of TGF-beta 1, was added in conjunction with IL-2 to previously stimulated B cell cultures, there was a 14.4 +/- 4.2% increase in B cell proliferation, a 22 +/- 6% increase in IgG production, and a 33 +/- 8.6% increase in IgM production when compared to control cultures. Chemical cross-linking of 125I-TGF-beta 1 to normal B cell membranes identified two major cross-linked species of 65 and 90 kDa. A fivefold excess of unlabeled TGF-beta 1 competitively inhibited the detection of both of these bands while a 50-fold excess of unlabeled TGF-beta 2 did not inhibit the 90-kDa band and only partially inhibited (60%) of the 65-kDa band. Chemical cross-linking of 125I-TGF-beta 1 to transformed B cell membranes identified only a single band of 60 kDa. Scatchard plot analysis of 125I-TGF-beta 1 binding to normal B cells that was competitively inhibited with increasing concentrations of unlabeled TGF-beta 1 revealed both high and low affinity binding sites whereas analysis of 125I-TGF-beta 1 binding in the presence of increasing concentrations of unlabeled TGF-beta 2 revealed only low affinity sites. These findings demonstrate that TGF-beta 2 is as effective as TGF-beta 1 in inhibiting human B cell function, that small amounts of active TGF-beta 1 are present endogenously in in vitro cultures which partially inhibit B cell function, that two major TGF-beta 1 R cross-linked complexes of 65 and 90 kDa are present on normal B cells, and that transformation of B cells may be accompanied by changes in the TGF-beta 1 R.     

Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-beta in vascular smooth muscle cells.

Previous studies have indicated that aged animals show an increased intimal hyperplasia after arterial injury. The present studies examined the hypothesis that the increased serum-free proliferation of aged smooth muscle cells (SMC), in vitro, was due to a loss of an antiproliferative signal, such as transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis of the mRNA derived from old (> 19 mo) or young (3-4 mo) rat aortic SMC indicated that both groups had an equivalent level of the 2.5 kB TGF-beta 1 message. Metabolic labeling with 35S-methionine and immunoprecipitation for TGF-beta 1 confirmed the de novo synthesis of TGF-beta 1 in rat SMC. Old and young SMC supernatants showed equal levels of active or latent (acid-activated) TGF-beta activity. Despite the similarities in the production of TGF-beta 1, old SMC were refractory to inhibition by TGF-beta 1, whereas young SMC were markedly inhibited (80%) by low levels of TGF-beta 1 (IC50 < 5 pg/ml). Binding studies at 4 degrees C indicated that old SMC exhibited reduced binding capacity for 125I-TGF-beta 1. Cross-linking studies confirmed that old SMC showed reduced binding of 125I-TGF-beta 1 to membrane sites corresponding to the high molecular weight type III receptor, as well as the 85-kDa type II and 65-kDa type I receptor. However, at 37 degrees C, old SMC degraded 125I-TGF-beta 1 more rapidly than young SMC. Combined, this data suggests that SMC derived from older animals are capable of normal production of TGF-beta 1 but fail to respond to the autocrine growth inhibitory effects of this agent, thereby leading to enhanced proliferation.