Cholesterol modulates cellular TGF-beta responsiveness by altering TGF-beta binding to TGF-beta receptors

Transforming growth factor-beta (TGF-beta) responsiveness in cultured cells can be modulated by TGF-beta partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. The TbetaR-II/TbetaR-I binding ratio of TGF-beta on the cell surface has recently been found to be a signal that controls TGF-beta partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF-beta binding to TGF-beta receptors and TGF-beta responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TbetaR-II/TbetaR-I binding ratio of TGF-beta while treatment with cholesterol-lowering or cholesterol-depleting agents increases the TbetaR-II/TbetaR-I binding ratio of TGF-beta in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TbetaR-II/TbetaR-I binding ratio of TGF-beta. Cholesterol treatment increases accumulation of the TGF-beta receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGF-beta responsiveness and statins/beta-CD enhances it, as measured by the levels of P-Smad2 and PAI-1 expression in cells stimulated with TGF-beta. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE-null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels may contribute to the pathogenesis of certain diseases (e.g., atherosclerosis) by suppressing TGF-beta responsiveness.     

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.     

The mannose 6-phosphate/insulin-like growth factor-II receptor is a substrate of type V transforming growth factor-beta receptor.

The type V transforming growth factor beta (TGF-beta) receptor (TbetaR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TbetaR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TbetaR-V in the presence of [gamma-32P]ATP and MnCl2 with an apparent Km of 130 nM. TGF-beta stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 degrees C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-beta stimulated Man-6-P/IGF-II receptor-mediated uptake (approximately 2-fold after 12 h treatment) of exogenous beta-glucuronidase in Mv1Lu cells and type II TGF-beta receptor (TbetaR-II)-defective mutant cells (DR26 cells) but not in type I TGF-beta receptor (TbetaR-I)-defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TbetaR-I and TbetaR-II but lacking TbetaR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitro and in vivo substrate of TbetaR-V and that both TbetaR-V and TbetaR-I may play a role in mediating the TGF-beta-stimulated uptake of exogenous beta-glucuronidase.     

TGF-beta control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.     

FK506, transforming growth factor-beta1 and mesangial matrix synthesis: parallels and differences compared with cyclosporine A

Cyclosporine A (CsA)-induced glomerulosclerosis is a well-described side effect of CsA treatment. Current evidence indicates that FK506 causes similar morphologic changes. Recently, we demonstrated that CsA up-regulates the expression of transforming growth factor-beta1 (TGF-beta1), its receptors type I (TbetaR-I) and type II (TbetaR-II), as well as related matrix protein synthesis in mesangial cells (MCs). Here, we assessed the effect of FK506 on the expression of TGF-beta1, TbetaR-I, TbetaR-II, fibronectin (FN) and plasminogen activator inhibitor type-1 (PAI-1) in MCs. Resting MCs were incubated with/without FK506. Time- and concentration-dependent expression was measured at the mRNA and protein level. Compared to untreated controls, FK506 stimulated TGF-beta1 mRNA (maximum at 8 h, 100 ng/mL: 2.13+/-0.15-fold, P<0.005) and protein expression (maximum at 96 h, 100 ng/mL: 1.96+/-0.29-fold, P<0.005). In contrast, TbetaR-I and TbetaR-II protein expression remained unchanged. Concerning matrix protein synthesis, FK506 slightly increased FN production (96 h, 100 ng/mL: 1.38+/-0.28-fold, P<0.05), but not PAI-1 production. These results indicate that, comparable to CsA, FK506 induced glomerulosclerosis is also due to a direct effect on mesangial matrix production, which is at least in part mediated via up-regulation of TGF-beta1 expression. The fact that, unlike CsA, FK506 does not increase the expression of TbetaR-I, TbetaR-II, and PAI-1, deserves further investigation.     

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.     

Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.     

The TGF-beta type III receptor is localized to the medial edge epithelium during palatal fusion

During palatal fusion, the medial edge epithelial cells (MEE) but not the oral/nasal palatal epithelium, selectively undergo epithelial-mesenchymal transformation. It is known that this process is regulated, at least in part, by endogenous TGF-beta3. One conceivable mechanism is that restricted expression of TGF-beta receptors (TbetaRs) in a subpopulation of cells may localize TGF-beta responsiveness (Brown et al., 1999). However, TGF-beta type II receptor (TbetaR-II) is expressed by all palatal epithelial cells during palatal fusion (Cui et al., 1998) and therefore cannot localize TGF-beta3 responsiveness. To investigate the role of TGF-beta type III receptor (TbetaR-III) in MEE transformation, we examined the expression pattern of TbetaR-III in the developing palate from E12 to E15 mice in vivo and in vitro by immunohistochemistry and compared the expression pattern to that of type I receptor (TbetaR-I). The expression of TbetaR-III was temporo-spatially restricted to the MEE during palatal fusion, while the expression of TbetaR-I was primarily localized in all palatal epithelia, consistent with the expression patterns of TbetaR-II and TGF-beta3 (Cui et al., 1998). These results support our hypothesis that TbetaR-III localizes and mediates the developmental role of TGF-beta3 on MEE transformation by specific expression in the MEE. TbetaR-III may modulate TGF-beta3 binding to TbetaR-II in the MEE cells to locally enhance TGF-beta3 autocrine signaling through the TbetaR-I/TbetaR-II receptor complex, which contributes to MEE selective epithelial-mesenchymal transformation.     

GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-beta receptors

Fibrosis around the smooth muscle of asthmatic airway walls leads to irreversible airway obstruction. Bronchial epithelial cells release granulocyte/macrophage colony-stimulating factor (GM-CSF) in asthmatics and are in close proximity to airway smooth muscle cells (ASMC). The findings in this study demonstrate that GM-CSF induces confluent, prolonged, serum-deprived cultures of ASMC to increase expression of collagen I and fibronectin. GM-CSF also induced ASMC to increase the expression of transforming growth factor (TGF)-beta receptors type I, II, and III (TbetaR-I, TbetaR-II, TbetaR-III), but had no detectable effect on the release of TGF-beta1 by the same ASMC. The presence of GM-CSF also induced the association of TGF-beta1 with TbetaR-III, which enhances binding of TGF-beta1 to TbetaR-II. The induction of TbetaRs was parallel to the increased induction of phosphorylated Smad2 (pSmad2) and connective tissue growth factor (CTGF), indicative of TGF-beta-mediated connective tissue synthesis. Dexamethasone decreased GM-CSF-induced TbetaR-I, TbetaR-II, TbetaR-III, pSmad2, CTGF, collagen I, and fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC to TGF-beta1-mediated connective tissue expression by induction of TbetaRs, which is inhibited by corticosteroids.     

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.     

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.     

Interactions of high affinity insulin-like growth factor-binding proteins with the type V transforming growth factor-beta receptor in mink lung epithelial cells

High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-beta receptor (TbetaR-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572-20576). To characterize the interactions of high affinity IGFBPs with TbetaR-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. 125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to TbetaR-V as demonstrated by the detection of the approximately 400-kDa 125I-IGFBP.TbetaR-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of 125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for TbetaR-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of 125I-IGFBP-3 preferentially bound to and was cross-linked to TbetaR-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-beta type I/type II receptor (TbetaR-I.TbetaR-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for TbetaR-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the TbetaR-I and TbetaR-II heterocomplex.     

Ontogeny and localization of TGF-beta type I receptor expression during lung development

Transforming growth factor (TGF)-beta is a family of multifunctional cytokines controlling cell growth, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-beta are mediated by type I (TbetaR-I) and II (TbetaR-II) receptors. Our previous studies show that the expression of TbetaR-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of TbetaR-I during lung development. The expression level of TbetaR-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. TbetaR-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of TbetaR-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. TbetaR-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense TbetaR-I signal was observed in the epithelial lining of the developing bronchi. In gestational day 16 lungs, the expression of TbetaR-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of TbetaR-I expression. In postnatal lungs, TbetaR-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of TbetaR-I in cultured rat lung cells. TbetaR-I was expressed by cultured rat lung fibroblasts, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of TbetaR-I that is different from that of TbetaR-II during lung development. TbetaR-I, TbetaR-II, and TGF-beta isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for TbetaR-I in mediating TGF-beta signal transduction during lung development.     

Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3-4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 microg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.     

Novel 150- and 180-kDa glycoproteins that bind transforming growth factor (TGF)-beta 1 but not TGF-beta 2 are present in several cell lines.

We identified transforming growth factor-beta (TGF-beta)-binding proteins which are distinct from previously described TGF-beta receptors or TGF-beta-binding proteins. These TGF-beta-binding proteins migrate as 150- and 180-kDa 125I-TGF-beta 1 affinity-labeled complexes which are consistently co-expressed in A549, Mv1Lu, MG-63, and BS-C-1 cells. They differ from the types I, II, and III TGF-beta receptors in their electrophoretic mobilities, their lack of binding to TGF-beta 2, and their failure to undergo the marked down-regulation seen with types I, II, and III receptors following a 16-h incubation with TGF-beta 1. The 150- and 180-kDa TGF-beta-binding proteins also are distinct from the recently described disulfide-linked TGF-beta 1-binding proteins which are present in rat glomeruli. In contrast to the glomerular TGF-beta 1-binding proteins, the electrophoretic mobilities of the 150- and 180-kDa binding proteins are unchanged following reduction. In addition, the 150- and 180-kDa TGF-beta-binding proteins are present in the detergent-rich phase during Triton X-114 phase separation, whereas the glomerular TGF-beta-binding proteins partition exclusively into the detergent-poor phase.     

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.     

Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms.

Characterization of the three mammalian transforming growth factor-beta (TGF-beta) isoforms, TGF-beta 1, -beta 2, and -beta 3, indicates that TGF-beta 3 is somewhat more potent (ED50 = 0.5 pM versus 2 pM) than TGF-beta 1 and TGF-beta 2 as a growth inhibitor of the Mv1Lu mink lung epithelial cell line. In the fetal bovine heart endothelial (FBHE) cell line, however, TGF-beta 1 and -beta 3 are at least 50-fold more potent than TGF-beta 2 which is a very weak growth inhibitor (ED50 greater than or equal to 0.5 nM). Thus, as growth inhibitors, TGF-beta 1 and -beta 3 resemble each other more than TGF-beta 2. The presence of serum alpha 2-macroglobulin in the FBHE cell assays decreases the biological potency of TGF-beta s, in particular TGF-beta 2. This effect of alpha 2-macroglobulin, however, is not sufficient to explain the low responsiveness of FBHE cells to TGF-beta 2. Evaluation of the role of TGF-beta receptors as determinants of cell-specific responsiveness to TGF-beta isoforms indicates that TGF-beta 1, -beta 2, and -beta 3 have similar affinity for the membrane proteoglycan, betaglycan. They differ, however, in their ability to bind to receptor types I and II which are implicated in TGF-beta signal transduction. TGF-beta 1 is similar, albeit not identical, to TGF-beta 3 and much more potent than TGF-beta 2 as a competitor for binding to the overall population of receptors I and II in all cell lines tested. A subset of receptors I and II has been identified in Mv1Lu cells which has high affinity for TGF-beta 2 (KD approximately 10 pM) and binds this factor at concentrations that are biologically active in Mv1Lu cells. This receptor subset could not be detected in FBHE cells, suggesting that cell-specific differences in the level of high affinity of TGF-beta 2 receptors may lead to cell-specific differences in responsiveness to this isoform. Thus, despite their structural and biological similarities, TGF-beta 1, -beta 2, and -beta 3 diverge in their ability to bind to receptors in a manner that correlates with their potency as growth inhibitors.