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.     

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.     

Influenza virus neuraminidase activates latent transforming growth factor beta.

Transforming growth factor beta (TGF-beta) is a family of proteins secreted by virtually all cells in a biologically inactive form. TGF-beta levels increase during many pathophysiological situations, including viral infection. The mechanism for increased TGF-beta activity during viral infection is not understood. We observed an increase in active TGF-beta levels within 1 day in mice infected with influenza virus. Further studies showed that the neuraminidase glycoprotein of influenza A and B viruses directly activates latent TGF-beta in vitro. There are sufficient levels of TGF-beta activated by virus to induce apoptosis in cells. In addition, influenza virus-induced apoptosis is partially inhibited by TGF-beta-specific antibodies. These novel findings suggest a potential role for activation of TGF-beta during the host response to influenza virus infection, specifically apoptosis. This is the first report showing direct activation of latent TGF-beta by a viral protein.     

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.     

The conformation-dependent interaction of alpha 2-macroglobulin with vascular endothelial growth factor. A novel mechanism of alpha 2-macroglobulin/growth factor binding

alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).     

Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance.

It has been widely assumed that the interaction of transforming growth factor-beta 1 (TGF-beta 1) with its serum-binding protein, alpha 2-macroglobulin (alpha 2M), mediates the rapid clearance of TGF-beta 1 from the circulation. To test this, we have analyzed the effect of TGF-beta 1 binding on the conformational state of alpha 2M. Our results demonstrate that the binding of TGF-beta 1 to alpha 2M does not lead to the conformational change in the alpha 2M molecule that is required for the clearance of the alpha 2M.TGF-beta 1 complex via the alpha 2M receptor. Furthermore, endogenous TGF-beta 1 is associated with the conformationally unaltered slow clearance form of alpha 2M. Clearance studies in mice show that the half-life of 125I-TGF-beta 1 in the circulation (1.6 +/- 0.71 min) is not affected by blocking the alpha 2M receptor with excess conformationally altered alpha 2M. These results suggest that TGF-beta 1 is rapidly cleared from the circulation after injection by a pathway not involving alpha 2M.     

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.     

A genetic screen to identify latent transforming growth factor beta activators

The mechanisms by which latent transforming growth factor beta (TGFbeta) is converted to the active cytokine are largely unknown. Here we present a genetic screen that combines retroviral mutagenesis and cDNA expression cloning to reveal proteins involved in the extracellular regulation of latent TGFbeta activation. The screen employs a cell line engineered to express green fluorescent protein (GFP) in response to TGFbeta. The cells produce their own latent TGFbeta. Therefore, after transduction with a retroviral cDNA library that contains an insert for an activator of latent TGFbeta, cells expressing the activator are GFP-bright. These cells are enriched by fluorescence-activated cell sorting and grown as individual clones. The isolated clones are cocultured with a second TGFbeta reporter cell line that produces luciferase in response to TGFbeta. Cells that have acquired the ability to activate latent TGFbeta induce luciferase expression in the absence but not in the presence of neutralizing antibodies to TGFbeta. The activator expressed by the positive clones can be identified by retrieval of the retrovirus cDNA insert.     

Anti-immunoglobulin treatment of murine B-cell lymphomas induces active transforming growth factor beta but pRB hypophosphorylation is transforming growth factor beta independent.

Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. When WEHI-231 lymphoma cells were selected for growth in TGF-beta, the majority of the TGF-beta-resistant clones remained sensitive to anti-Ig-mediated growth inhibition. In these clones, the retinoblastoma gene product was found to be in the underphosphorylated form after 24-h treatment with anti-Ig, but not with TGF-beta. These data show that anti-Ig treatment of murine B-cell lymphomas stimulates the production of active TGF-beta but that a TGF-beta-independent pathway may be responsible for the pRB underphosphorylation and cell cycle blockade.     

Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.     

Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as a latent high molecular weight complex in a human erythroleukemia cell line, HEL, treated with phorbol 12-myristate 13-acetate. The complex is comprised of three components: mature TGF-beta 1, the TGF-beta 1 latency-associated peptide (beta 1-LAP), and the latent TGF-beta 1-binding protein (LTBP). LTBP plays an important role in the assembly and secretion of the latent TGF-beta 1 complex; if the TGF-beta 1 precursor fails to bind to LTBP, much of it remains inside the cells and may contain anomalous disulfide bond(s) between beta 1-LAP and the mature TGF-beta 1 molecule (Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991) EMBO J. 10, 1091-1101). In the present work, we have investigated the subcellular localization and properties of the TGF-beta 1 precursor retained intracellularly. When the HEL cells were metabolically labeled and chased for up to 72 h, a considerable part of the TGF-beta 1 precursor was still observed intracellularly in an unprocessed form. The secreted form of the TGF-beta 1 precursor was resistant to endoglycosidase H, whereas the intracellular form of the TGF-beta 1 precursor was sensitive to endoglycosidase H, regardless of the presence or absence of swainsonine, an inhibitor of mannosidase II. Indirect immunofluorescence microscopy revealed that the TGF-beta 1 precursor co-localized with mannosidase II, a marker for the Golgi complex, but not with protein disulfide isomerase, a marker for the endoplasmic reticulum. The intracellular TGF-beta 1 precursor was prepared from phorbol 12-myristate 13-acetate-treated HEL cells and tested for TGF-beta 1 bioactivity. Half-maximal inhibition of the DNA synthesis in mink lung epithelial cells, Mv1Lu, was observed at 80 pM of the acid-treated TGF-beta 1 precursor, whereas nontreated material showed minimal growth inhibitory activity. Taken together, these results indicate that the TGF-beta 1 precursor is retained inside the cells in the Golgi complex, mainly in a latent, immature form.     

Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha.

The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.     

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*.     

Functional analysis of cathepsin B-like cysteine proteases from Leishmania donovani complex. Evidence for the activation of latent transforming growth factor beta

Cathepsin B-like genes from Leishmania donovani and Leishmania chagasi have been isolated and characterized. It is a single gene, which is constitutively expressed in all the life cycle stages of the parasite. Studies using cathepsin B-specific inhibitor treatment suggested that cathepsin B does not seem to play a role in the promastigote stages of the parasite, however it aids in the parasite survival within the host macrophages. Antisense mRNA inhibition of cathepsin B gene also revealed that it plays an important role in the parasite survival within the host macrophages. Furthermore, for the first time, we have shown that Leishmania whole cell lysates as well as the recombinant cathepsin B protein cleaved human recombinant latent transforming growth factor (TGF)-beta1 into a mature peptide releasing the latency associated protein, in a cell-free incubation system. Mink lung epithelial cell growth inhibition assay revealed that the cleaved TGF-beta1 was biologically active, suggesting that Leishmania cathepsin B can cleave latent TGF-beta1 into mature and active form. These results suggest that cathepsin B plays an important role in Leishmania survival within the host macrophages by activating latent TGF-beta1.     

Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha

Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate-polyacrylamide gels, the apparent molecular weight of these intracellular TGF-alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF-beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate-polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta.     

Normal embryo fibroblasts release transforming growth factors in a latent form

Normal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.     

Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification

Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.