Purification and properties of epithelial growth inhibitor (EGI) from human platelets: its separation from type beta transforming growth factor (TGF-beta)

We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major component of type beta transforming growth factor (TGF-beta), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27,000, and was composed of two subunits identical in Mr. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium containing 10% fetal calf serum. Its inhibitory activity was stable against heating at 90 degrees C for 3 min, but not against treatment with 50 mM dithiothreitol. In addition, TGF-beta was also partially purified from the same extract. The purified TGF-beta did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK.     

Cripto binds transforming growth factor beta (TGF-beta) and inhibits TGF-beta signaling

Cripto is a developmental oncoprotein and a member of the epidermal growth factor-Cripto, FRL-1, Cryptic family of extracellular signaling molecules. In addition to having essential functions during embryogenesis, Cripto is highly expressed in tumors and promotes tumorigenesis. During development, Cripto acts as an obligate coreceptor for transforming growth factor beta (TGF-beta) ligands, including nodals, growth and differentiation factor 1 (GDF1), and GDF3. As an oncogene, Cripto is thought to promote tumor growth via mechanisms including activation of mitogenic signaling pathways and antagonism of activin signaling. Here, we provide evidence supporting a novel mechanism in which Cripto inhibits the tumor suppressor function of TGF-beta. Cripto bound TGF-beta and reduced the association of TGF-beta with its type I receptor, TbetaRI. Consistent with its ability to block receptor assembly, Cripto suppressed TGF-beta signaling in multiple cell types and diminished the cytostatic effects of TGF-beta in mammary epithelial cells. Furthermore, targeted disruption of Cripto expression by use of small inhibitory RNA enhanced TGF-beta signaling, indicating that endogenous Cripto plays a role in restraining TGF-beta responses.     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.     

IGG-stimulated and LPS-stimulated monocytes elaborate transforming growth factor type beta (TGF-beta) in active form

Mononuclear cells (MNC) stimulated either with lipopolysaccharide (LPS) or with surface-adsorbed IgG elaborated significant amounts of tumor necrosis factor (TNF) bioactivity, as well as immunoenzymatically detectable TNF-alpha and interleukin-1 beta. (IL1-beta). In contrast, IgG-stimulated cells released little IL1 bioactivity, but released an IL1 inhibitor, as determined by the thymocyte costimulatory assay (LAF assay). This inhibition was not due to an inhibitory effect of cyclooxygenase products, e.g. prostaglandin-E2 in the LAF assay. In contrast, antibodies against transforming growth factor type beta (TGF-beta), which is an important inhibitor of the LAF assay, augmented the LAF activity of supernatants from LPS-stimulated and IgG-stimulated MNC. Anti-TGF-beta-modulated LAF inhibition was enhanced by acid treatment of supernatants from mononuclear cells, but not of those from purified monocytes. Antibody blocking experiments point for the first time to a TGF-beta species other than type 1 as a monocyte-derived TGF-beta activity. Thus, TGF-beta released in active form from monocytes may be the more important antagonist of IL1 than cyclooxygenase-derived mediators. It implies that the LAF assay, in the absence of anti-TGF-beta antibodies, is an inadequate indicator of IL1 activity.     

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.     

Regulation of type 1 diabetes by a self-MHC class II peptide: role of transforming growth factor beta (TGF-beta).

The present study was undertaken to analyze the regulatory T cells generated in response to class I derived self-I-A beta(g7) (54-76) peptide. It was observed T cells from young unprimed type 1 diabetes (T1D) prone NOD mice did not respond to self-I-A beta(g7) (54-76) peptide although T cells from primed young NOD mice showed a strong response. T cells from young unprimed BALB/c mice responded to self-I-A beta(d) (62-78) peptide. However, a breakdown of tolerance to these peptides was observed with age in both the strains. Culture supernatant from I-A beta(g7) (54-76) peptide-primed cells secreted large amounts of TGF-beta and inhibited T cell responses in allogeneic-MLR. Further, I-A beta(g7) (54-76) peptide specific T cell lines from young (I-A.Y) and diabetic (I-A.D) NOD mice were established. I-A.Y secreted IL-4, TGF-beta and IL-10 while I-A.D T cell line secreted IL-10 and IFN-gamma. We found that I-A.D T cell line induced diabetes when transferred in NOD/SCID mice but I-A.Y T cell line did not induce disease. These results show that immunization of NOD mice with I-A beta(g7) (54-76) peptide at a younger age induces a regulatory T cell response suggesting that correcting the defects in immunoregulatory mechanisms using self-MHC peptides may be one of the approaches to prevent autoimmune diseases like T1D.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro

Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.     

Expression of functional Schistosoma mansoni Smad4: role in Erk-mediated transforming growth factor beta (TGF-beta) down-regulation

Members of the transforming growth factor (TGF)-beta superfamily play pivotal roles in cell migration, differentiation, adhesion, pattern formation, and apoptosis. The family of Smad proteins acts as intracellular signal transducers of TGF-beta and related peptides. Smad4, a common mediator Smad (co-Smad), performs a central role in transmitting signals from TGF-beta, BMP, and activins. Schistosoma mansoni receptor-regulated Smad1 and SmSmad2 were previously identified and shown to act in TGF-beta signaling. Herein, we report the identification and characterization of a Smad4 homologue from S. mansoni and provide details about its role in mediation and down-regulation of TGF-beta signaling in schistosomes. In order to identify the schistosome co-Smad, we designed degenerate primers based on the sequence of the conserved MH1/MH2 domains of Smad4 proteins, which were used in PCR to amplify a 137-bp PCR product. A S. mansoni adult worm pair cDNA library was screened resulting in the isolation of a cDNA clone that encodes a 738 amino acid protein (SmSmad4). SmSmad4 was shown to interact with schistosome R-Smads (SmSmad1 and SmSmad2) in vivo and in vitro. The interaction with SmSmad2 was dependent on the receptor-mediated phosphorylation of SmSmad2. In addition, several potential phosphorylation sites for Erk1/2 kinases were identified in the SmSmad4 linker region and shown to be phosphorylated in vitro by an active mutant of mammalian Erk2. Furthermore, Erk-mediated phosphorylation of SmSmad4 decreased its interaction with the receptor-activated form of SmSmad2, in vitro. SmSmad4 was shown to complement a human Smad4 deficiency through the restoration of TGF-beta-responsiveness in MDA-MB-468 breast cancer cells.     

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.     

Differential response of cultured rabbit articular chondrocytes (RAC) to transforming growth factor beta (TGF-beta)-evidence for a role of serum factors

We show that addition of TGF-beta (0.01-10 ng/ml) to proliferating rabbit articular chondrocytes in presence of low level of fetal calf serum (FCS, 2%) results in a sustained decrease of cell number and DNA synthesis up to 72 h. In contrast, incubation with high serum concentration (10% FCS) induces a transient increase of cell number after 48 h without elevation of DNA synthesis. Moreover, when the factor is added in 10% FCS-containing medium, a differential effect is observed at 48 h (either increase or decrease of cell number) depending on the serum level (2 or 10%) present between 24 and 48 h. Recruitment of cells in late S-phase occurred under TGF-beta-treatment in both 2 and 10% FCS. These arrested cells may then be released by further exposure to 10% FCS-containing medium. The data show that factor(s) from the serum modulate(s) the action of TGF-beta on chondrocyte proliferation. Addition of epidermal growth factor (EGF) to the cultures in presence of 2% FCS mimicks the effects observed with 10% serum, suggesting that the serum component(s) involved in the mechanism could be of EGF type.     

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.     

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration

Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions.     

Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta.

Regulation of the human type 1 plasminogen activator inhibitor (PAI-1) promoter by transforming growth factor-beta (TGF beta) was studied. An 800-base pair fragment from the PAI-1 promoter and 5'-flanking region was fused to the firefly luciferase reporter gene and transfected into Hep3B human hepatoma cells. Treatment of the cells with TGF beta induced luciferase activity by more than 50-fold. Transfection studies using constructs with 5' or 3' deletions through this region revealed that two sequences were important in the TGF beta response. The first sequence was located in the proximal promoter (-49 to -87) and mediated an 11-fold induction with TGF beta, while the second more distal region (-636 to -740) contained two sequences which together mediated a 50-fold or greater response. Sequence comparison indicated that both of the responsive regions contained sequences with high homology to the AP-1 consensus binding site. Moreover, gel retardation analysis experiments demonstrated that both sequences bound a common nuclear protein, and that an oligonucleotide containing a consensus AP-1 sequence was able to compete for the binding of this common protein. Thus, the response of the PAI-1 gene to TGF beta is mediated by at least two separate regions, and both of these regions contain DNA sequences homologous to the AP-1 binding site.     

Transforming growth factor beta (TGF-beta) type V receptor has a TGF-beta-stimulated serine/threonine-specific autophosphorylation activity.

The transforming growth factor beta (TGF-beta) type V receptor, a newly identified high molecular weight TGF-beta receptor (M(r) approximately 400,000) has been purified from bovine liver plasma membranes (O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 266, 8583-8589). The purified TGF-beta type V receptor underwent autophosphorylation at serine residues when incubated with [gamma-32P]ATP in the presence of 0.1% beta-mercaptoethanol and 2.5 mM MnCl2. This phosphorylation was stimulated by preincubation with TGF-beta. The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. The TGF-beta type V receptor could be affinity-labeled with 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine. Polylysine appeared to stimulate the autophosphorylation of the TGF-beta type receptor in the presence of [gamma-32P]ATP and the incorporation of 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine into the TGF-beta type V receptor. The amino acid sequence analysis of the peptide fragments produced by cyanogen bromide cleavage of the purified TGF-beta type V receptor revealed that a peptide, namely CNBr-19, contained an amino acid sequence which shows homology to the putative ATP binding site of the receptors for activin, the Caenorhabditis elegans daf-1 gene product, and TGF-beta type II receptor (Lin, H. Y., Wang, Y.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785). These results suggest that the TGF-beta type V receptor is a Ser/Thr-specific protein kinase and belongs to the new class of membrane receptors associated with a Ser/Thr-specific protein kinase activity.