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.     

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-beta receptor profiles of human and murine embryonic palate mesenchymal cells.

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-beta (TGF-beta) class of growth factors is suggested by the immunolocalization of TGF-beta 1 in the palate during its ontogeny. This study examined the effects of TGF-beta stimulation of, as well as TGF-beta receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-beta 1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-beta 1 binding to specific receptors was determined by incubating cells in the presence of 170 pM 125I-TGF-beta 1 for up to 4 h. In both cell types, at 37 degrees C, the binding of 125I-TGF-beta decreased linearly over 4 h, while at 4 degrees C, binding increased with time of incubation. Incubation of both cell types at 4 degrees C for 4 h, with increasing concentrations of 125I-TGF-beta 1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 pM) and MEPM (K 26.3 pM). However, SDS-PAGE analyses of 125I-TGF-beta chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 Mr) and III (230,000 Mr) TGF-beta receptors while MEPM also contained the type II (86,000 Mr) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-beta binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-beta and have disparate TGF-beta receptor profiles.     

Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin

The biological latency of serum transforming growth factor-beta (TGF-beta) was shown to be due to the interaction of TGF-beta with a specific serum binding protein. This binding protein was affinity labeled with 125I-TGF-beta, and its Mr and subunit structure were determined using sodium dodecyl sulfate-gel electrophoresis and gel filtration chromatography. Its Mr is reminiscent of that of the serum protease inhibitor, alpha 2-macroglobulin (alpha 2M). Immunoprecipitation of the 125I-TGF-beta-binding protein complex by a specific anti-alpha 2M antibody, and the formation of identical complexes between 125I-TGF-beta and purified alpha 2M, confirmed that alpha 2M is the TGF-beta-binding protein in serum. Immunoblot analysis showed that endogenous serum TGF-beta is also bound to alpha 2M. However, in contrast to added 125I-TGF-beta, the majority of the endogenous TGF-beta is linked to alpha 2M covalently. Alpha 2M and acid-activated TGF-beta co-eluted from a Superose 6 fast protein liquid chromatography column, confirming that the interaction of TGF-beta with alpha 2M accounts for the latency of serum TGF-beta. It is proposed that alpha 2M may serve an important multifunctional role at sites of inflammation by scavenging both active peptides and proteases that are released by platelets at the site of injury.     

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.     

Identification of disulfide-linked transforming growth factor-beta 1-specific binding proteins in rat glomeruli

We have identified two distinct classes of transforming growth factor-beta (TGF-beta)-binding proteins by affinity labeling rat glomeruli with 125I-TGF-beta 1 and 125I-TGF-beta 2. The first type consists of a group of proteins that bind TGF-beta 1 but do not bind TGF-beta 2. When 125I-TGF-beta 1 affinity-labeled glomeruli were separated under nonreducing conditions, four prominent bands with Mr values of 320,000, 260,000, 170,000, and 90,000 were observed. Following reduction, the 320,000 and 170,000 bands yielded only a 100,000 band, the 260,000 complex yielded bands of 200,000, 100,000, and 85,000, and the 90,000 band migrated with an Mr of 85,000. Binding of 125I-TGF-beta 1 to these proteins was unaffected by the addition of as much as a 1,000-fold excess of TGF-beta 2. The second type of glomerular TGF-beta-binding protein consists of Mr 160,000-200,000 and 280,000 proteins that bind both TGF-beta 1 and beta 2. Digestion of these affinity-labeled proteins with heparitinase and chondroitinase resulted in a decrease of approximately 40,000 in their apparent molecular weights. Glomerular TGF-beta 1-binding proteins are distinct from previously described TGF-beta-binding proteins in their specificity for TGF-beta 1 and their formation of disulfide-linked multimers. The TGF-beta 1/beta 2-binding proteins share some properties of the previously described type III TGF-beta receptor.     

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.     

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.     

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.     

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.     

Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1

The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.     

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.     

Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

Differential inhibition of transforming growth factor beta 1 and beta 2 activity by alpha 2-macroglobulin.

Affinity labeling and immunoprecipitation studies demonstrate that alpha 2-macroglobulin (alpha 2M) is the major serum-binding protein for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2). Purified alpha 2M inhibits the binding of both 125I-TGF-beta 1 and 125I-TGF-beta 2 to cell surface receptors at I50 values of 200 and 10 micrograms/ml, respectively. alpha 2M (200 micrograms/ml) does not block TGF-beta 1 inhibition of CCL-64 mink lung cell growth but reduces this activity of TGF-beta 2 10-fold. The electrophoretic migration of 125I-TGF-beta.alpha 2M complexes on polyacrylamide gels under nondenaturing conditions demonstrates that alpha 2M has 10-fold greater affinity for TGF-beta 2 than for TGF-beta 1. Each of these complexes comigrates as a single band with the fast form of alpha 2M. We suggest that alpha 2M is an important differential regulator of the biological activities of TGF-beta 1 and TGF-beta 2 in vivo.     

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.     

Transforming growth factor-beta inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes.

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-beta (TGF-beta). In this paper, we show that TGF-beta inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-beta-treated chondrocytes show reduced 125I-IL-1 binding that returns to a normal level when TGF-beta is removed from the culture medium. The inhibitory effect of TGF-beta is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor-ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of Mr = 116 and 80 kDa and a minor band of Mr = 100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-beta treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-beta to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

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.