SMART MODE:

NORMAL GENOMIC

• Harpel JG, Schultz-Cherry S, Murphy-Ullrich JE, Rifkin DB
• Tamoxifen and estrogen effects on TGF-beta formation: role of thrombospondin-1, alphavbeta3, and integrin-associated protein.
• Biochem Biophys Res Commun. 2001; 284: 11-4
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• We have found that the enhanced activation of latent TGF-beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavbeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-beta activation. Copyright 2001 Academic Press.

• Unsold C, Hyytiainen M, Bruckner-Tuderman L, Keski-Oja J
• Latent TGF-beta binding protein LTBP-1 contains three potential extracellular matrix interacting domains.
• J Cell Sci. 2001; 114: 187-197
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• Latent TGF-beta binding proteins (LTBPs) are components of the extracellular matrix (ECM). They belong to the fibrillin/LTBP-superfamily, and are high molecular weight glycoproteins characterized by EGF-like repeats and 8-Cys repeats. Most LTBPs associate with the small latent forms of TGF-beta. Their roles include to facilitate the secretion of latent TGF-beta and to target it to the ECM. In order to identify new matrix-binding domains of LTBP-1 and to characterize their association with the extracellular matrix, we have produced (in a mammalian expression system) partly overlapping recombinant fragments of its shorter form, LTBP-1S, and analyzed the binding of the purified fusion proteins to extracellular matrices of cultured human dermal and lung fibroblasts. Recombinant fragments from three different regions of the N- and C-termini showed affinity to the matrix. These interacting regions contain either the first (hybrid), second or fourth 8-Cys domains of the LTBP-1S molecule. They bound independently to the matrix. Each of them had an ability to inhibit the association of native exogenous LTBP-1 with fibroblast extracellular matrix. The interactions of the LTBP-1 fragments with the extracellular matrix resisted treatment with sodium deoxycholate, suggesting strong, possibly covalent binding. The binding occurred in a time- and dose-dependent fashion. The N-terminal fragments bound more readily to the matrices. With all fragments the binding took place both with intact fibroblast matrices and with matrices isolated by sodium deoxycholate. When using CHO cell layers, which form sparse matrices, only the N-terminal fragment of LTBP-1 was efficiently incorporated. The association of the binding fragments with isolated matrices was enhanced by soluble, cell-derived factors. The current data suggest that LTBP-1 contains three different domains with an ability to associate with the extracellular matrix.

• Sasaki A, Naganuma H, Satoh E, Kawataki T, Amagasaki K, Nukui H
• Participation of thrombospondin-1 in the activation of latent transforming growth factor-beta in malignant glioma cells.
• Neurol Med Chir (Tokyo). 2001; 41: 253-8
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• Malignant glioma cells secrete transforming growth factor-beta (TGF-beta) and can activate latent TGF-beta. However, the mechanism of the latent TGF-beta activation has not yet been determined. This study examined whether thrombospondin-1 (TSP-1) secreted by malignant glioma cell lines participates in the activation of latent TGF-beta secreted by the glioma cells. Western blot analysis revealed that TSP-1 was present in both the cell lysates and the culture supernatants of all three malignant glioma cell lines (T98G, A172, and U251). A bioassay for TGF-beta activity revealed that all malignant glioma cell lines used in this study could activate latent TGF-beta by themselves. Latent TGF-beta 1 activation, evaluated by enzyme-linked immunosorbent assay, was inhibited by more than 50% by the addition of neutralizing anti-TSP-1 monoclonal antibody or anti-TSP-1 polyclonal antibody. These results indicate that TSP-1 has a predominant role in the activation of latent TGF-beta in malignant glioma cells.

• Laing KJ, Cunningham C, Secombes CJ
• Genes for three different isoforms of transforming growth factor-beta are present in plaice (Pleuronectes platessa) DNA.
• Fish Shellfish Immunol. 2000; 10: 261-71
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• Although transforming growth factor-beta (TGF-beta) genes have been described in several species of fish, whether an individual fish possesses more than one member of this multigene family has yet to be established. During this study, three DNA fragments were isolated from the plaice (Pleuronectes platessa) by homology cloning. Sequence analysis revealed that each fragment closely resembled a distinct member of the TGF-beta family. Each putative plaice TGF-beta clustered individually with a different TGF-beta subgroup during phylogenetic analysis suggesting that these may be the plaice homologues of vertebrate TGF-beta 1/4/5, -beta 2 or -beta 3. The first direct evidence for the presence of multiple TGF-beta genes in a single fish species is presented.

• Tsumura H, Ishii Y, Shimizu E, Ohashi H, Mori KJ
• Generation of recombinant human large latent transforming growth factor-beta 1 and monoclonal antibodies to it.
• Biosci Biotechnol Biochem. 2000; 64: 17-23
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• Transforming growth factor beta 1 (TGF-beta 1) is a regulator of cell growth and differentiation. It is produced in various of cells and tissues as a biologically latent complex, whose significance is still unknown. We established a Chinese hamster ovary cells that produced recombinant human large latent TGF-beta 1. The growth factor was purified from serum-free conditioned medium of the cell line was purified to apparent homogeneity by four steps of column chromatography. The purified protein gave a single band with the apparent molecular weight of 210,000 on SDS-PAGE, and had four subunits, of 12.5, 40, 53, and 150-190 kDa. These components were identical to TGF-beta 1, the N-terminal remnant of pro-TGF-beta 1, pro-TGF-beta 1, and latent TGF-beta 1 binding protein, respectively. The purified growth factor had biological activity similar to that of the growth factor purified from human platelets. We prepared four monoclonal antibodies by immunization of mice with the recombinant protein. In western blotting, two of the antibodies bound to latent TGF-beta 1 binding protein. The two other antibodies reacted with the N-terminal remnant of pro-TGF-beta 1. Recombinant large latent TGF-beta 1 and its monoclonal antibodies could be used for detailed structural and functional studies of the large latent TGF-beta 1 complex.

• Murphy-Ullrich JE, Poczatek M
• Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology.
• Cytokine Growth Factor Rev. 2000; 11: 59-69
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• Regulation of the activation of latent TGF-beta is essential for health as too much or too little TGF-beta activity can have serious, deleterious consequences. The processes that control conversion of the precursor to the biologically active form of TGF-beta in vivo are not well characterized. We have identified a mechanism for the activation of latent TGF-beta that involves binding of the secreted and extracellular matrix protein, thrombospondin-1 (TSP-1), to the latent precursor. Specific sequences in TSP-1 and in the precursor portion (the latency associate peptide-LAP) have been determined to be essential for activation of latent TGF-beta by TSP-1. It is thought that binding of TSP-1 to the latent complex induces a conformational rearrangement of the LAP in such a manner as to prevent the LAP from conferring latency on the mature domain of TGF-beta. A TSP-dependent mechanism of activation may be locally important during wound healing and in post-natal development of epithelial structures. The possible involvement of TSP-1 in TGF-beta activation during several disease processes is also discussed.

• Poczatek MH, Hugo C, Darley-Usmar V, Murphy-Ullrich JE
• Glucose stimulation of transforming growth factor-beta bioactivity in mesangial cells is mediated by thrombospondin-1.
• Am J Pathol. 2000; 157: 1353-63
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• Glucose is a key factor in the development of diabetic complications, including diabetic nephropathy. The development of diabetic glomerulosclerosis is dependent on the fibrogenic growth factor, transforming growth factor-beta (TGF-beta). Previously we showed that thrombospondin-1 (TSP-1) activates latent TGF-beta both in vitro and in vivo. Activation occurs as the result of specific interactions of latent TGF-beta with TSP-1, which potentially alter the conformation of latent TGF-beta. As glucose also up-regulates TSP-1 expression, we hypothesized that the increased TGF-beta bioactivity observed in rat and human mesangial cells cultured with high glucose concentrations is the result of latent TGF-beta activation by autocrine TSP-1. Glucose-induced bioactivity of TGF-beta in mesangial cell cultures was reduced to basal levels by peptides from two different sequences that antagonize activation of latent TGF-beta by TSP, but not by the plasmin inhibitor, aprotinin. Furthermore, glucose-dependent stimulation of matrix protein synthesis was inhibited by these antagonist peptides. These studies demonstrate that glucose stimulation of TGF-beta activity and the resultant matrix protein synthesis are dependent on the action of autocrine TSP-1 to convert latent TGF-beta to its biologically active form. These data suggest that antagonists of TSP-dependent TGF-beta activation may be the basis of novel therapeutic approaches for ameliorating diabetic renal fibrosis.

• Huang SS, Zhou M, Johnson FE, Shieh HS, Huang JS
• An active site of transforming growth factor-beta(1) for growth inhibition and stimulation.
• J Biol Chem. 1999; 274: 27754-8
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• Transforming growth factor-beta (TGF-beta) is a bifunctional growth regulator. It inhibits growth of many cell types, including epithelial cells, but stimulates growth of others (e.g. fibroblasts). The active site on the TGF-beta molecule, which mediates its growth regulatory activity, has not been defined. Here, we show that antibody to a TGF-beta(1) peptide containing the motif WSLD (52nd to 55th amino acid residues) completely blocked both (125)I-TGF-beta(1) binding to TGF-beta receptors and TGF-beta(1)-induced growth inhibition in mink lung epithelial cells. Site-directed mutagenesis analysis revealed that the replacement of Trp(52) and Asp(55) by alanine residues diminished the growth inhibitory activity of TGF-beta(1) by approximately 90%. Finally, while wild-type TGF-beta(1) was able to stimulate growth of transfected NIH 3T3 cells, the double mutant TGF-beta(1) W52A/D55A was much less active. These results support the hypothesis that the WSLD motif is an active site of TGF-beta(1), which is important for growth inhibition of epithelial cells and growth stimulation of fibroblasts.

• Saharinen J, Hyytiainen M, Taipale J, Keski-Oja J
• Latent transforming growth factor-beta binding proteins (LTBPs)--structural extracellular matrix proteins for targeting TGF-beta action.
• Cytokine Growth Factor Rev. 1999; 10: 99-117
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• Growth factors of the transforming growth factor-beta family are potent regulators of the extracellular matrix formation, in addition to their immunomodulatory and regulatory roles for cell growth. TGF-beta s are secreted from cells as latent complexes containing TGF-beta and its propeptide, LAP (latency-associated peptide). In most cells LAP is covalently linked to an additional protein, latent TGF-beta binding protein (LTBP), forming the large latent complex. LTBPs are required for efficient secretion and correct folding of TGF-beta s. The secreted large latent complexes associate covalently with the extracellular matrix via the N-termini of the LTBPs. LTBPs belong to the fibrillin-LTBP family of extracellular matrix proteins, which have a typical repeated domain structure consisting mostly of epidermal growth factor (EGF)-like repeats and characteristic eight cysteine (8-Cys) repeats. Currently four different LTBPs and two fibrillins have been identified. LTBPs contain multiple proteinase sensitive sites, providing means to solubilize the large latent complex from the extracellular matrix structures. LTBPs are now known to exist both as soluble molecules and in association with the extracellular matrix. An important consequence of this is LTBP-mediated deposition and targeting of latent, activatable TGF-beta into extracellular matrices and connective tissues. LTBPs have a dual function, they are required both for the secretion of the small latent TGF-beta complex as well as directing bound latent TGF-beta to extracellular matrix microfibrils. However, it is not known at present whether LTBPs are capable of forming microfibrils independently, or whether they are a part of the fibrillin-containing fibrils. Most LTBPs possess RGD-sequences, which may have a role in their interactions with the cell surface. At least LTBP-1 is chemotactic to smooth muscle cells, and is involved in vascular remodelling. Analyses of the expressed LTBPs have revealed considerable variations throughout the molecules, generated both by alternative splicing and utilization of multiple promoter regions. The significance of this structural diversity is mostly unclear at present.

• Ribeiro SM, Poczatek M, Schultz-Cherry S, Villain M, Murphy-Ullrich JE
• The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.
• J Biol Chem. 1999; 274: 13586-93
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• One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.

• Gleizes PE, Rifkin DB
• [Activation of latent TGF-beta. A required mechanism for vascular integrity]
• Pathol Biol (Paris). 1999; 47: 322-9
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• Recent molecular genetics studies in humans and mice showed that transforming growth factor-beta (TGF-beta) is involved in vasculogenesis and maintenance of blood vessel integrity. These results confirm earlier in vitro studies demonstrating generation of active TGF-beta when endothelial cells are cocultured with smooth muscle cells or pericytes. TGF-beta is secreted as a latent, inactive complex and becomes active only when released. Latent TGF-beta binds covalently to proteins (LTBP) that target it to the extracellular matrix. Thus, the latency of TGF-beta is essential to the regulation of the bioavailability and activity of this cytokine. The development of methods for measuring activation of latent TGF-beta in cell cultures and identification of the proteins contained in the latent TGF-beta complex have shed new light on the mechanism of activation of latent TGF-beta possibly involved in vasculogenesis, angiogenesis, and other processes.

• Chong H, Vodovotz Y, Cox GW, Barcellos-Hoff MH
• Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages.
• J Cell Physiol. 1999; 178: 275-83
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• Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

• Munger JS, Harpel JG, Giancotti FG, Rifkin DB
• Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1.
• Mol Biol Cell. 1998; 9: 2627-38
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• The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-beta function in the ECM, we determined whether latent TGF-beta1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits alphav and beta1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways.

• Roth S, Michel K, Gressner AM
• (Latent) transforming growth factor beta in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat hepatic stellate cells.
• Hepatology. 1998; 27: 1003-12
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• Cultured parenchymal liver cells (PC) were recently recognized to contain (latent) transforming growth factor beta (TGF-beta) while the expression of TGF-beta mRNA remains controversial. This study was designed to analyze PC in different microenvironments (liver in situ, highly purified, isolated, and cultured PC) regarding the qualitative and quantitative content of mature and latent TGF-beta protein (immunostainings, enzyme-linked immunosorbent assay [ELISA], and enzyme-labeled fluorescence [ELF] technique). The results were compared with its gene expression (reverse-transcription polymerase chain reaction [RT-PCR]). In all microenvironments, PC contained latent TGF-beta, which was partially activated after cell isolation and culture. The amount of total TGF-beta (mature plus latent) of latency-associated peptide (LAP) and of latent TGF-beta binding protein (LTBP) were shown to decrease during culture. In contrast, TGF-beta2 and TGF-beta3 mRNA and LTBP-1 and -3 mRNA expression were first detectable after culture. Permeabilization of cell membranes in whole liver and of isolated PC with streptolysin O or carbon tetrachloride, respectively, released TGF-beta, a part of which was integrated in the large latent complex as estimated by analytical gel filtration chromatography. The TGF-beta released by damaged PC induces paracrine effects on hepatic stellate cell cultures. It stimulates hyaluronan synthesis and antagonizes the effect of mitogenic factor(s) of PC on [3H]thymidine incorporation. The results strongly suggest that the main part of hepatocellular TGF-beta is not generated by de novo synthesis but from uptake into the liver in vivo. The immunodetection of preexisting mature TGF-beta after isolation of the cells is probably caused by intracellular activation of latent TGF-beta. The injury-dependent discharge of TGF-beta from PC might be an important mechanism for initiation and perpetuation of various forms of chronic human liver diseases.

• Carey GB, Chang NS
• Cloning and characterization of a transforming growth factor beta 1-induced anti-apoptotic adhesion protein TIF2.
• Biochem Biophys Res Commun. 1998; 249: 283-6
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• Transforming growth factor-beta (TGF-beta) antagonizes the cytotoxic function of tumor necrosis factor (TNF). By differential display and library screening, we isolated a murine TIF2 (TGF-beta-induced factor 2) cDNA, encoding a putative 15-kDa membrane adhesion protein, which possesses an RGD sequence at the extracellular region. When TNF-sensitive murine L929 fibroblasts were stably transfected with TIF2 cDNA, these cells significantly resisted TNF killing. In contrast, L929 cells, which stably expressed the TIF2 antisense mRNA, acquired enhanced TNF susceptibility. Calculated EC50 values, i.e., the amount of TNF needed for killing 50% cells, are 10, 55, and 1.5 ng/ml, respectively, for vector control, sense transfectant, and antisense transfectant. TGF-beta 1 rapidly induces TIF2 gene expression (approximately 1 hr), which correlates with time-related acquisition of TNF-resistance in TGF-beta 1-treated L929 cells. Notably, TIF2 gene expression is markedly increased in human breast cancer and lymphoid leukemia cells, compared to normal human cells, suggesting its potential role in cancer development. Together, the anti-apoptotic function of TIF2 is responsible in part for TGF-beta-mediated protection of L929 cells against TNF cytotoxicity.

• Letterio JJ, Roberts AB
• Regulation of immune responses by TGF-beta.
• Annu Rev Immunol. 1998; 16: 137-61
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• The transforming growth factor beta (TGF-beta) family of proteins are a set of pleiotropic secreted signaling molecules with unique and potent immunoregulatory properties. TGF-beta 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. TGF-beta can modulate expression of adhesion molecules, provide a chemotactic gradient for leukocytes and other cells participating in an inflammatory response, and inhibit them once they have become activated. Increased production and activation of latent TGF-beta have been linked to immune defects associated with malignancy and autoimmune disorders, to susceptibility to opportunistic infection, and to the fibrotic complications associated with chronic inflammatory conditions. In addition to these roles in disease pathogenesis, TGF-beta is now established as a principal mediator of oral tolerance and can be recognized as the sine qua non of a unique subset of effector cells that are induced in this process. The accumulated knowledge gained through extensive in vitro functional analyses and from in vivo animal models, including newly established TGF-beta gene knockout and transgenic mice, supports the concept that clinical therapies based on modulation of this cytokine represent an important new approach to the treatment of disorders of immune function.

• Taipale J, Saharinen J, Keski-Oja J
• Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion.
• Adv Cancer Res. 1998; 75: 87-134
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• Growth factors of the transforming growth factor-beta (TGF-beta) family inhibit the proliferation of epithelial, endothelial, and hematopoietic cells, and stimulate the synthesis of extracellular matrix components. TGF-beta s are secreted from cells in high-molecular-mass protein complexes that are composed of three proteins, the mature TGF-beta-dimer, the TGF-beta propeptide dimer, or latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Mature TGF-beta is cleaved from its propeptide during secretion, but the proteins remain associated by noncovalent interactions. LTBP is required for efficient secretion and processing of latent TGF-beta and it binds to LAP via disulfide bond(s). LTBP is a component of extracellular matrix microfibrils, and it targets the latent TGF-beta complex to the extracellular matrix. TGF-beta signaling is initiated by proteolytic cleavage of LTBP that results in the release of the latent TGF-beta complex from the extracellular matrix. TGF-beta is activated by dissociation of LAP from the mature TGF-beta. Subsequent signaling involves binding of active TGF-beta to its type II cell surface receptors, which phosphorylate and activate type I TGF-beta receptors. Type I receptors, in turn, phosphorylate cytoplasmic transcriptional activator proteins Smad2 and Smad3, inducing their translocation to the nucleus. Recent evidence suggests that acquisition of resistance to TGF-beta growth inhibition plays a major role in the progression of epithelial and hematopoietic cell malignancies. The role of secretion of TGF-beta in tumorigenesis is more complex. The secretion of TGF-beta s by tumor cells may contribute to autocrine growth inhibition, but on the other hand, it may also promote invasion, metastasis, angiogenesis, and even immunosuppression. Tumor cells may also fail to deposit LTBP:TGF-beta complexes to the extracellular matrix. The elucidation of the mechanisms of the release of TGF-beta from the matrix and its subsequent activation aids the understanding of the pathophysiologic roles of TGF-beta in malignant growth, and allows the development of therapeutic agents that regulate the activity of TGF-beta.

• Gong W, Roth S, Michel K, Gressner AM
• Isoforms and splice variant of transforming growth factor beta-binding protein in rat hepatic stellate cells.
• Gastroenterology. 1998; 114: 352-63
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• BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are one important source for transforming growth factor beta (TGF-beta). They produce TGF-beta in a latent form associated with latency-associated peptide and latent TGF-beta-binding protein (LTBP). This study was designed to investigate, on RNA and protein levels, which isoforms of LTBP and TGF-beta are expressed in HSCs and myofibroblasts. METHODS: HSCs isolated from rat liver were analyzed for LTBP and TGF-beta at various times of culture during transdifferentiation into myofibroblasts using immunocytochemical staining, metabolic labeling and immunoprecipitation, reverse-transcription polymerase chain reaction (RT-PCR), and sequencing. RESULTS: Alkaline phosphatase-anti-alkaline phosphatase staining and fluorescence immunostainings indicated the expression of all three components of the large latent TGF-beta complex in HSCs and myofibroblasts. Transcripts of three TGF-beta and LTBP isoforms were detected by RT-PCR and confirmed by sequence analyses. A new LTBP-1 splice form was found lacking part of the hinge region with a potential proteinase cleavage site. Metabolic labeling followed by immunoprecipitation with LTBP antiserum confirmed the synthesis and secretion of various LTBP-related proteins. CONCLUSIONS: The existence of different LTBP isoforms and splice variants in HSCs and myofibroblasts suggests multiple functions of the LTBP family in rat liver, which might not be restricted to the maintenance of TGF-beta latency.

• Abe M, Oda N, Sato Y
• Cell-associated activation of latent transforming growth factor-beta by calpain.
• J Cell Physiol. 1998; 174: 186-93
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• Transforming growth factor-beta (TGF-beta) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-beta complex that makes the complex inactive, activates latent TGF-beta. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-beta. When recombinant latent TGF-beta was incubated with calpain (1-10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-beta from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05-0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-beta antibody as well as by calpain inhibitor peptide, but not by alpha2-antiplasmin, a specific inhibitor of plasmin. Active TGF-beta was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of (125)I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-beta through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-beta, which is targeted to the same surface.

• Nunes I et al.
• Structure and activation of the large latent transforming growth factor-Beta complex.
• J Am Optom Assoc. 1998; 69: 643-8
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• BACKGROUND: Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-beta (TGF-beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS: A review of experimental results that demonstrate how vascular cells generate biologically active TGF-beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-beta as a large latent TGF-beta complex. Mature TGF-beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS: A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-beta. CONCLUSION: The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation. Studying TGF-beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-beta activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies.

• Haworth RS, Brooks G, Cummins P, Dobie K, Chilton DC, Avkiran M
• Differential changes in transforming growth factor-beta isoform expression during postnatal cardiac growth.
• Biochem Biophys Res Commun. 1998; 245: 923-7
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• Transforming growth factor-beta (TGF-beta) is synthesised as an inactive precursor protein; this is cleaved to produce the mature peptide and a latency associated protein (LAP), which remains associated with the mature peptide until activation by LAP degradation. Isoform specific antibodies raised against the LAPs for TGF-beta 2 and -beta 3 were used to determine the myocardial levels of LAP (activatable TGF-beta) and full length precursor (inactive TGF-beta) forms during post-natal development in the rat. TGF-beta 2 was present predominantly as the precursor in 2 day old myocardium. There was an age-dependent shift from precursor protein to LAP between 2 and 28 days. A corresponding increase in the level of mature (activatable) TGF-beta 2 was found. TGF-beta 3 was detected in significant quantities only as LAP. However, a four-fold increase in the expression of TGF-beta 3 LAP was observed between 2 and 28 days. The substantial increases in activatable forms of TGF-beta 2 and -beta 3 that occur in myocardium during the first 28 days of life in the rat support a role for these proteins in post-natal cardiac development.

• Reiss M, Barcellos-Hoff MH
• Transforming growth factor-beta in breast cancer: a working hypothesis.
• Breast Cancer Res Treat. 1997; 45: 81-95
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• Transforming Growth Factor-beta (TGF beta) is the most potent known inhibitor of the progression of normal mammary epithelial cells through the cell cycle. During the early stages of breast cancer development, the transformed epithelial cells appear to still be sensitive to TGF beta-mediated growth arrest, and TGF beta can act as an anti-tumor promoter. In contrast, advanced breast cancers are mostly refractory to TGF beta-mediated growth inhibition and produce large amounts of TGF beta, which may enhance tumor cell invasion and metastasis by its effects on extracellular matrix. We postulate that this seemingly paradoxical switch in the responsiveness of tumor cells to TGF beta during progression is the consequence of the activation of the latent TGF beta that is produced and deposited into the tumor microenvironment, thereby driving the clonal expansion of TGF beta-resistant tumor cells. While tumor cells themselves may activate TGF beta, recent observations suggest that environmental tumor promoters or carcinogens, such as ionizing radiation, can cause stromal fibroblasts to activate TGF beta by epigenetic mechanisms. As the biological effects of the anti-estrogen tamoxifen may well be mediated by TGF beta, this model has a number of important implications for the clinical uses of tamoxifen in the prevention and treatment of breast cancer. In addition, it suggests a number of novel approaches to the treatment of advanced breast cancer.

• Guo NH, Krutzsch HC, Inman JK, Shannon CS, Roberts DD
• Antiproliferative and antitumor activities of D-reverse peptides derived from the second type-1 repeat of thrombospondin-1.
• J Pept Res. 1997; 50: 210-21
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• The extracellular matrix glycoprotein thrombospondin-1 (TSP1) inhibits angiogenesis, endothelial cell growth, motility and adhesion. Peptides from the type I repeats of TSP1 mimic the adhesive and growth inhibitory activities of the intact protein and specifically interact with heparin and transforming growth factor-beta (TGF beta). To define the structural basis for the antiangiogenic activities of these peptides, we prepared analogs of the TSP1 peptide KRFKQDGGWSHWSPWSSC. L-forward, L-reverse, and D-reverse (retro-inverso) analogs displayed identical activities for binding to heparin, demonstrating a lack of stereospecificity for heparin binding. The L-reverse and D-reverse peptides, however, had somewhat decreased abilities to activate latent TGF beta. Conjugation of the forward peptides through a C-terminal thioether and the reverse peptides through an N-terminal thioether to polysucrose abolished the adhesive activity of the peptides and enhanced their antiproliferative activities for endothelial and breast carcinoma cells stimulated by fibroblast growth factor-2. Their antiproliferative activities were independent of latent TGF beta activation, because substitution of an Ala residue for the essential Phe residue in the TSP1 type-1 repeat peptide increased their potency for inhibiting TSP1 binding to heparin and for inhibiting endothelial cell proliferation. Although the conjugated peptides were inactive in vivo, an unconjugated retro-inverso analog of the native TSP peptide inhibited breast tumor growth in a mouse xenograft model. Thus, these TSP-derived peptide analogs antagonize endothelial growth through their heparin-binding activity rather than through activation of latent TGF beta or increasing cell adhesion. These stable analogs may therefore be useful as therapeutic inhibitors of angiogenesis stimulated by fibroblast growth factor-2.

• Yuan X, Downing AK, Knott V, Handford PA
• Solution structure of the transforming growth factor beta-binding protein-like module, a domain associated with matrix fibrils.
• EMBO J. 1997; 16: 6659-66
• Display abstract

• Here we describe the high resolution nuclear magnetic resonance (NMR) structure of a transforming growth factor beta (TGF-beta)-binding protein-like (TB) domain, which comes from human fibrillin-1, the protein defective in the Marfan syndrome (MFS). This domain is found in fibrillins and latent TGF-beta-binding proteins (LTBPs) which are localized to fibrillar structures in the extracellular matrix. The TB domain manifests a novel fold which is globular and comprises six antiparallel beta-strands and two alpha-helices. An unusual cysteine triplet conserved in the sequences of TB domains is localized to the hydrophobic core, at the C-terminus of an alpha-helix. The structure is stabilized by four disulfide bonds which pair in a 1-3, 2-6, 4-7, 5-8 pattern, two of which are solvent exposed. Analyses of MFS-causing mutations and the fibrillin-1 cell-binding RGD site provide the first clues to the surface specificity of TB domain interactions. Modelling of a homologous TB domain from LTBP-1 (residues 1018-1080) suggests that hydrophobic contacts may play a role in its interaction with the TGF-beta1 latency-associated peptide.

• Nagai K, Ishizuka I
• [Mammalian adhesive proteins that bind to sulfated glycolipids]
• Seikagaku. 1997; 69: 1186-91

• Penha-Goncalves MN, Onions DE, Nicolson L
• Cloning and sequencing of equine transforming growth factor-beta 1 (TGF beta-1) cDNA.
• DNA Seq. 1997; 7: 375-8
• Display abstract

• Transforming growth factor beta (TGF-beta) belongs to a family of peptide growth factors which control critical stages of cell proliferation and differentiation. We report the cloning and sequencing of the cDNA for TGF-beta type 1 isoform of the horse. The predicted mature equine TGF beta-1 peptide is 112 amino acids in length and exhibits 99% identity to mature human TGF beta-1.

• Bonewald LF, Oreffo RO, Lee CH, Park-Snyder S, Twardzik D, Mundy GR
• Effects of retinol on activation of latent transforming growth factor-beta by isolated osteoclasts.
• Endocrinology. 1997; 138: 657-66
• Display abstract

• The multifunctional cytokine, transforming growth factor-beta (TGF beta), is found in many tissues in a latent or inactive form. The nature and composition of the latent complex can vary depending on tissue type. The release of active TGF beta from its latent complex is a potentially important mechanism for regulation of TGF beta activity. We have shown previously that osteoclasts activate latent TGF beta produced by bone and that bone cells produce a 100-kDa latent complex that lacks the latent TGF beta-binding protein. Here we investigated the effects of retinol on osteoclast activation of various forms of latent TGF beta. Two sources of osteoclasts were used that provide either mature avian osteoclasts or avian osteoclast precursors. Whereas both cell populations activate latent TGF beta, only mature osteoclasts respond to retinol with an increase in activation of latent TGF beta over basal levels. Activation could not be ascribed to pH changes in conditioned medium. Nonacid-dissociable 100-kDa latent complex, which is also produced by bone cells, was added to mature osteoclasts and to osteoclast precursors, but no activation was observed. Platelet latent TGF beta, which contains the 130-kDa latent TGF beta-binding protein, was activated by both osteoclast populations. Conditioned medium from the precursor population activated latent complex, whereas conditioned medium from mature cells did not. Activation of latent TGF beta by retinol-treated mature cells was not blocked by inhibitors of plasmin, nor was activation by conditioned medium from precursor cells. These data suggest that retinol-induced activation of latent TGF beta by osteoclasts is dependent on the stage of differentiation of these cells and the presence of other cell types, and that unlike other cell systems, the plasmin-plasminogen activator mechanism is not involved.

• Munger JS, Harpel JG, Gleizes PE, Mazzieri R, Nunes I, Rifkin DB
• Latent transforming growth factor-beta: structural features and mechanisms of activation.
• Kidney Int. 1997; 51: 1376-82
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• Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGF-beta s are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-beta. LAP is disulfide linked to another protein, latent TGF-beta binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF-beta to the matrix. Activation of latent TGF-beta can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF-beta is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF-beta is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin.

• Huang SS, Liu Q, Johnson FE, Konish Y, Huang JS
• Transforming growth factor beta peptide antagonists and their conversion to partial agonists.
• J Biol Chem. 1997; 272: 27155-9
• Display abstract

• Transforming growth factor beta (TGF-beta) has been implicated in the pathogenesis of various human diseases. Synthetic TGF-beta antagonists therefore could have therapeutic utility. Here we show the development of such compounds. Three synthetic pentacosapeptides designated beta125-(41-65), beta225-(41-65), and beta325-(41-65), whose amino acid sequences correspond to the 41st to 65th amino acid residues of TGF-beta1, TGF-beta2, and TGF-beta3, respectively, inhibit the binding of 125I-labeled TGF-beta isoforms to TGF-beta receptors in mink lung epithelial cells with IC50 of approximately 0.06-2 microM. beta125-(41-65) blocks TGF-beta1-induced growth inhibition and TGF-beta1-induced plasminogen activator inhibitor-1 expression in these cells. The variants designated beta125-(41-65)W52A/D55A and beta325-(41-65)R52A/D55A, in which both Trp52/Arg52 and Asp55 are replaced by alanine residues, do not have TGF-beta antagonist activity. Multiple conjugation of beta125-(41-65) to carrier proteins enhances its antagonist activity but also confers partial agonist activity as measured by DNA synthesis inhibition. These results suggest that the (W/R)XXD motif is important for the activities of these TGF-beta peptide antagonists and that this motif may be the active site sequence of TGF-beta.

• Piek E, Franzen P, Heldin CH, ten Dijke P
• Characterization of a 60-kDa cell surface-associated transforming growth factor-beta binding protein that can interfere with transforming growth factor-beta receptor binding.
• J Cell Physiol. 1997; 173: 447-59
• Display abstract

• We have characterized a 60-kDa transforming growth factor-beta (TGF-beta) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross-linking of cell surface proteins by using 125I-TGF-beta 1. Binding of 125I-TGF-beta 1 to the 60-kDa protein was competed by an excess of unlabeled TGF-beta 1 but not by TGF-beta 2, TGF-beta 3, activin, or osteogenic protein-1 (OP-1), also termed bone morphogenetic protein-7 (BMP-7). In addition, no binding of 125I-TGF-beta 2 and 125I-TGF-beta 3 to the 60-kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60-kDa TGF-beta binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF-beta superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 microgram/ml heparin, or 10% glycerol caused a release of the 60-kDa protein from the cell surface. In addition, we found that the previously described TGF-beta type IV receptor on GH3 cells, which does not form a heterometric complex with TGF-beta receptors, could be released from the cell surface by these same treatments. This suggests that the 60-kDa protein and the similarly sized TGF-beta type IV receptor are related proteins. The eluted 60-kDa LNCaP protein was shown to interfere with the binding of TGF-beta to the TGF-beta receptors. Thus, the cell surface-associated 60-kDa TGF-beta binding protein may play a role in regulating TGF-beta binding to TGF-beta receptors.

• Gleizes PE, Beavis RC, Mazzieri R, Shen B, Rifkin DB
• Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1.
• J Biol Chem. 1996; 271: 29891-6
• Display abstract

• Most cultured cell types secrete small latent transforming growth factor-beta (TGF-beta) as a disulfide-bonded complex with a member of the latent TGF-beta binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-beta1. Coexpression in Sf9 cells of small latent TGF-beta1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-beta1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-beta, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-beta1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-beta.

• Marra F, Bonewald LF, Park-Snyder S, Park IS, Woodruff KA, Abboud HE
• Characterization and regulation of the latent transforming growth factor-beta complex secreted by vascular pericytes.
• J Cell Physiol. 1996; 166: 537-46
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• Transforming growth factor-beta (TGF-beta) stimulates the accumulation of extracellular matrix in renal and hepatic disease. Kidney glomerular mesangial cells (GMC) and liver fat-storing cells (FSC) produce latent of inactive TGF-beta. In this study, we characterized the latent TGF-beta complexes secreted by these cells. Human FSC produce a single latent TGF-beta complex, predominantly of the TGF-beta 1 isoform, whereas GMC secrete multiple complexes of latent TGF-beta, containing beta 1 and beta 2 isoforms. At least four forms were identified in GMC using ion exchange chromatography, including a peak not previously described in other cell types which eluted at 0.12 M NaCl, and predominantly of the beta 2 isoform. Both cell types secrete the latent TGF-beta 1 binding protein of 190 kDa, as part of a high molecular weight TGF-beta complex. Epidermal growth factor stimulates the secretion of latent TGF-beta and latent TGF-beta binding protein in both cell types. Secretion of latent TGF-beta in both cell types was found to be associated with secretion of decorin. This study shows that vascular pericytes from the kidney and the liver have distinctly different profiles of latent TGF-beta complexes, with GMC secreting a unique form of latent TGF-beta 2. The regulatory effect of epidermal growth factor and platelet-derived growth factor has potential implication for the pathophysiology of liver regeneration and chronic liver and kidney diseases.

• Qian SW et al.
• Binding affinity of transforming growth factor-beta for its type II receptor is determined by the C-terminal region of the molecule.
• J Biol Chem. 1996; 271: 30656-62
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• Transforming growth factor-beta (TGF-beta) isoforms have differential binding affinities for the TGF-beta type II receptor (TbetaRII). In most cells, TGF-beta1 and TGF-beta3 bind to TbetaRII with much higher affinity than TGF-beta2. Here, we report an analysis of the effect of TGF-beta structure on its binding to TbetaRII by using TGF-beta mutants with domain deletions, amino acid replacements, and isoform chimeras. Examination of the binding of TGF-beta mutants to the recombinant extracellular domain of TbetaRII by a solid-phase TGF-beta/TbetaRII assay demonstrated that only those TGF-beta mutants containing the C terminus of TGF-beta1 (TGF-beta1-(Delta69-73), TGF-beta1-(Trp71), and TGF-beta2/beta1-(83-112)) bind with high affinity to TbetaRII, similar to native TGF-beta1. Moreover, replacement of only 6 amino acids in the C terminus of TGF-beta1 with the corresponding sequence of TGF-beta2 (TGF-beta1/beta2-(91-96)) completely eliminated the high affinity binding of TGF-beta1. Proliferation of fetal bovine heart endothelial (FBHE) cells was inhibited to a similar degree by all of the TGF-beta mutants. However, recombinant soluble TbetaRII blocked the inhibition of FBHE cell proliferation induced by TGF-beta mutants retaining the C terminus of TGF-beta1, consistent with the high binding affinity between these TGF-beta molecules and TbetaRII. It was further confirmed that the TGF-beta2 mutant with its C terminus replaced by that of TGF-beta1 (TGF-beta2/beta1-(83-112)) competed as effectively as TGF-beta1 with 125I-TGF-beta1 for binding to membrane TbetaRI and TbetaRII on FBHE cells. These observations clearly indicate that the domain in TGF-beta1 responsible for its high affinity binding to TbetaRII, both the soluble and membrane-bound forms, is located at C terminus of the molecule.

• Ghosh S, Brauer PR
• Latent transforming growth factor-beta is present in the extracellular matrix of embryonic hearts in situ.
• Dev Dyn. 1996; 205: 126-34
• Display abstract

• Transforming growth factor-beta (TGF-beta) is an important regulator of development. In vitro, TGF-beta is secreted in a latent, inactive form and can be activated by pH extremes, chaotropic agents, or cell-surface proteases. However, there is little evidence for the existence of latent TGF-beta in vivo. In this study, we determined whether (1) cultured embryonic cardiac segments secrete latent or active TGF-beta, (2) binding of TGF-beta antibody to TGF-beta was conformation-dependent (i.e., active vs. latent), and (3) immunostaining of embryonic hearts changed after exposure to activating conditions. Only latent TGF-beta 3 (acid activatable) was detected in conditioned medium of stage 14-16 chick cardiac segments as measured by a growth inhibition bioassay. No growth-inhibitory activity was present in nonacidified control medium. When blotted onto a membrane, only transiently acidified conditioned medium bound TGF-beta antibody. These data showed that cardiac segments secrete latent TGF-beta which binds with antibody if activated. To determine if antibody binding to tissue sections required exposure to TGF-beta-activating conditions, stage 14-16 embryos were fixed and sectioned under conditions that maximally retained extracellular matrix (ECM). Under these conditions, immunostaining was found in the myocardium but not in the endocardium or cardiac ECM. Limited immunostaining was found in other areas of the embryo and was always cell-associated. In addition to the above staining, when tissue sections were exposed to TGF-beta activating conditions, immunopositive staining was present within most of the embryonic ECM including the cardiac ECM. All immunostaining was blocked by preabsorption with TGF-beta 3 protein. These data suggest that active TGF-beta has a very limited distribution while latent TGF-beta is more abundant in embryonic ECM. Therefore, in vivo activation of TGF-beta may play an important role in mediating the expression of TGF-beta function during development.

• Rogers ML, Goddard C, Regester GO, Ballard FJ, Belford DA
• Transforming growth factor beta in bovine milk: concentration, stability and molecular mass forms.
• J Endocrinol. 1996; 151: 77-86
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• Transforming growth factor beta (TGF-beta) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-beta in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a milk lung epithelial cell line. Most of the TGF-beta bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-beta concentration was 4.3 +/- 0.8 ng and 3.7 +/- 0.7 ng TGF-beta per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6.5 was used to concentrate latent whey-derived TGF-beta, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-beta. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-beta with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-beta from both high molecular mass peaks. Whey-derived TGF-beta was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-beta 2, which accounted for over 85% of the TGF-beta bioactivity in whey. All TGF-beta activity in whey could be neutralised by a monoclonal antibody directed against TGF-beta 1, -beta 2 and -beta 3. The results suggest that the majority of TGF-beta in bovine milk is present in a small latent complex.

• Saharinen J, Taipale J, Keski-Oja J
• Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1.
• EMBO J. 1996; 15: 245-53
• Display abstract

• Transforming growth factor-betas (TGF-betas) are produced by most cells in large latent complexes of TGF-beta and its propeptide (LAP) associated with a binding protein. The latent TGF-beta binding proteins (LTBPs-1, -2 and -3) mediate the secretion and, subsequently, the association of latent TGF-beta complexes with the extracellular matrix (ECM). The association of beta1-LAP with LTBP-1 was characterized at the molecular level with an expression system in mammalian cells, where TGF-beta1 and various fragments of LTBP-1 were co-expressed and secreted with the aid of a signal peptide synthesized to the LTBP-1 constructs. Immunoblotting of the fusion protein complexes indicated that the third 8-Cys repeat of LTBP-1 bound covalently to the LAP region of TGF-beta1. The cysteine required for the association between LTBP-1 and beta1-LAP was mapped to Cys33 of beta1-LAP. The N-terminal region of LTBP-1 consisting of the first 400 amino acids was found to associate covalently with the ECM. The data indicate that an 8-Cys repeat of LTBP is capable of covalent and specific protein-protein interactions. These interactions are mediated by exchanging cysteine disulfide bonds between the core 8-Cys repeat and an optionally associated protein during the secretion. This is, to our knowledge, the first demonstration of an extracellular protein module that is able to exchange cysteine disulfide bonds with heterologous ligand proteins.

• Schultz-Cherry S, Hinshaw VS
• Influenza virus neuraminidase activates latent transforming growth factor beta.
• J Virol. 1996; 70: 8624-9
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• 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.

• Ogasa H, Noma T, Murata H, Kawai S, Nakazawa A
• Cloning of a cDNA encoding the human transforming growth factor-beta type II receptor: heterogeneity of the mRNA.
• Gene. 1996; 181: 185-90
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• We have isolated two novel cDNAs encoding the transforming growth factor-beta (TGF-beta) type II receptor (TGF-beta IIR), termed TGF-beta IIR alpha and TGF-beta IIR beta 1 from a human fetal liver library. They have unique nucleotide (nt) sequences, compared with the reported TGF-beta IIR sequence, at the 5' end. Southern blot analysis using probes from each clone detected the specific genomic DNA fragments. RT-PCR analysis revealed a distinct pattern of expression for each isoform. These results indicated that TGF-beta IIR has heterogeneity in the structure, and the expression of TGF-beta IIR isoforms is differentially regulated. The heterogeneity of TGF-beta IIR molecules could be derived from alternative splicing and might elicit specific TGF-beta receptor functions.

• Olofsson A, Ichijo H, Moren A, ten Dijke P, Miyazono K, Heldin CH
• Efficient association of an amino-terminally extended form of human latent transforming growth factor-beta binding protein with the extracellular matrix.
• J Biol Chem. 1995; 270: 31294-7
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• Latent transforming growth factor-beta (TGF-beta) binding protein-1 (LTBP-1) is a component of the high molecular weight latent TGF-beta complex found in various cells, including human platelets. LTBP-1 is observed as different molecular sizes in different cell types, probably due to proteolytic processing and alternative splicing. We here report a novel form of human LTBP-1, which is longer in its NH2-terminal part (LTBP-1L). Northern hybridization analysis revealed that the LTBP-1L is derived from a 7.0-kilobase mRNA, whereas the originally reported shorter form (LTBP-1S) is derived from a 5.2-kilobase mRNA. Transfection of cDNA for LTBP-1L and -1S in COS cells revealed that LTBP-1L bound more efficiently to the extracellular matrix than did LTBP-1S. These results suggest that the different splice forms of LTBP-1 mediate different localization patterns of the latent TGF-beta complexes in vivo.

• Wang TN, Qian XH, Granick MS, Solomon MP, Rothman VL, Tuszynski GP
• The effect of thrombospondin on oral squamous carcinoma cell invasion of collagen.
• Am J Surg. 1995; 170: 502-5
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• BACKGROUND: Thrombospondin (TSP), a cell matrix protein, and transforming growth factor beta (TGF-beta), a growth regulatory protein, play roles in tumor progression. The purpose of this study was to investigate the effects of TSP and TGF-beta on tumor cell invasion. MATERIALS AND METHODS: Tumor cell invasion assays were performed using a modified Boyden chamber apparatus with collagen-coated membranes. The KB oral carcinoma cell line was studied in serum-free media. Invasion was measured as the summation of the number of cells in five representative low-power fields (x 100) traversing the collagen barrier after a 3-hour incubation period. The effects of antibodies against TSP, TGF-beta and the cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG)-specific TSP receptor were also evaluated. RESULTS: TSP caused a dose-dependent stimulation of tumor cell invasion. Antibodies against TSP, its CSVTCG-specific receptor, and TGF-beta inhibited TSP-promoted invasion by 50% to 71%. CONCLUSIONS: TSP and its CSVTCG-specific receptor promote KB cell invasion of collagen through the production and/or activation of TGF-beta.

• Souchelnitskiy S, Chambaz EM, Feige JJ
• Thrombospondins selectively activate one of the two latent forms of transforming growth factor-beta present in adrenocortical cell-conditioned medium.
• Endocrinology. 1995; 136: 5118-26
• Display abstract

• Transforming growth factor-beta (TGF beta) has been shown previously to be a potent inhibitor of bovine adrenocortical cell steroidogenic functions. However, it is present in the culture medium of these cells in a latent form. In this study, we analyzed in detail the biochemical composition of this latent TGF beta. Two distinct complexes could be separated chromatographically by gel filtration on Sephacryl S-300, and their composition was studied using immunochemical methods. The results indicate that one form (peak I) is a complex between alpha 2-macroglobulin (alpha 2M) and either the unprocessed TGF beta precursor or the mature form of TGF beta. In a major fraction of this complex, TGF beta is covalently linked to alpha 2 M, whereas in a minor fraction, it is noncovalently bound and, therefore, activatable. The second form of latent TGF beta (peak II) is a complex among latent TGF beta-binding protein (LTBP), latency-associated protein, and mature TGF beta and a complex between LTBP and unprocessed TGF beta. We investigated the ability of thrombospondins (TSP1 and TSP2) to activate these latent forms of TGF beta. TSP1 and TSP2 were equally potent at activating the LTBP-latency-associated protein-TGF beta complex in the absence of cell contact, but were ineffective on the alpha 2M-TGF beta complex. Therefore, TGF beta may act as an autocrine regulator of adrenocortical steroidogenic functions. Its activity appears to be controlled by TSPs, the local production of which is regulated by systemic ACTH.

• Adams JC, Lawler J
• Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.
• Mol Biol Cell. 1994; 5: 423-37
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• Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family.

• Taipale J, Miyazono K, Heldin CH, Keski-Oja J
• Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein.
• J Cell Biol. 1994; 124: 171-81
• Display abstract

• The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse-chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.

• Oursler MJ
• Osteoclast synthesis and secretion and activation of latent transforming growth factor beta.
• J Bone Miner Res. 1994; 9: 443-52
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• The coupling of bone resorption and formation suggests that autocrine and paracrine factors are produced and released within the local bone environment. Since osteoblasts secrete latent transforming growth factor beta (TGF-beta), and osteoclasts can activate this latent complex, one candidate paracrine coupling factor is TGF-beta. To examine whether TGF-beta is also a potential osteoclast-derived autocrine and paracrine factor, highly purified avian osteoclasts were examined for synthesis of TGF-beta and the mechanism by which osteoclasts activate the latent TGF-beta complex was investigated. TGF-beta protein production was measured within 4 h of culture in osteoclast-conditioned media using growth factor inhibition of CCL-64 cells and verified by blocking effects with anti-TGF-beta antibodies. Synthesis of TGF-beta was confirmed by northern blotting and metabolic labeling. Northern blots of total RNA revealed that osteoclasts expressed the mRNA for TGF-beta 2, beta 3, and beta 4. Biosynthetic studies suggest that TGF-beta 2 was the principle form secreted in culture. Nearly all the TGF-beta that was secreted had been activated by the osteoclasts. When presented with exogenous latent TGF-beta, osteoclasts activated latent TGF-beta from a variety of sources. Furthermore, osteoclast-conditioned media retained the ability to activate latent TGF-beta during cell-free incubations at 37 degrees C. Osteoclast-mediated activation was inhibited by proteinase inhibitors, weak base treatment of the cells, or disruption of the cytoskeletal network. These data suggest that osteoclasts may secrete proteinases into the extracellular milieu to activate latent TGF-beta and that TGF-beta may be an autocrine factor involved in regulating osteoclast activity.

• McCartney-Francis NL, Wahl SM
• Transforming growth factor beta: a matter of life and death.
• J Leukoc Biol. 1994; 55: 401-9
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• A wide variety of functions, many of which represent opposing activities, have been attributed to TGF-beta, a molecule implicated in embryogenesis, development, and immune and inflammatory processes. This paradoxical behavior of promoting or inhibiting cell growth and function, while important in normal physiology and homeostasis, can contribute to or interrupt pathologic sequelae, making TGF-beta a particularly intriguing molecule for study. New transgenic mouse models displaying targeted alterations in TGF-beta 1 expression offer novel and unique opportunities to determine the essential function(s) of TGF-beta.

• Bassing CH, Howe DJ, Segarini PR, Donahoe PK, Wang XF
• A single heteromeric receptor complex is sufficient to mediate biological effects of transforming growth factor-beta ligands.
• J Biol Chem. 1994; 269: 14861-4
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• Transforming growth factor beta (TGF-beta), a multifunctional cytokine that regulates a variety of biological functions, signals through a heteromeric receptor complex of the type I and type II TGF-beta receptors. The type II receptor, a transmembrane serine-threonine kinase, was cloned based on its ability to directly bind TGF-beta. Recently, a number of candidate type I TGF-beta receptors have been isolated. Although only one of these transmembrane kinases (R4) has been shown to mediate TGF-beta-dependent gene activation, others bind TGF-beta when overexpressed in COS cells. Consequently, it has been postulated that the diversity of TGF-beta responses is generated through the association of distinct type I receptors with the type II TGF-beta receptor, thus creating receptor complexes of differential signaling capacities. In contrast to this model, we demonstrate that stable expression of only the R4 type I TGF-beta receptor in a mutant cell line lacking endogenous type I TGF-beta receptor was able to complex with the endogenous type II TGF-beta receptor and restore the effects of TGF-beta on inhibition of cell proliferation and activation of specific genes, regardless of which of the three mammalian isoforms of TGF-beta was used as the ligand. Therefore, R4 acts as a fully functional type I TGF-beta receptor, and the differential effects of TGF-beta are likely mediated by a single receptor complex consisting of R4 and the type II receptor.

• Schultz-Cherry S, Ribeiro S, Gentry L, Murphy-Ullrich JE
• Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system.
• J Biol Chem. 1994; 269: 26775-82
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• Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein normally secreted by cells in a latent form. Primary regulation of TGF-beta activity occurs through factors which control the processing of the latent to the biologically active molecule. Thrombospondin (TSP), a platelet alpha-granule and extracellular matrix protein, forms specific complexes with active TGF-beta in platelet releasate and activates endogenous latent TGF-beta secreted by endothelial cells via a cell- and protease-independent mechanism. In order to better understand TSP-mediated activation of cell-secreted latent TGF-beta, we examined the consequences of interactions of the large (platelet-derived) and small (recombinant) forms of latent TGF-beta with TSP in a chemically defined system. Data from these studies show that interactions between TSP and both forms of latent TGF-beta result in the generation of biologically active TGF-beta as assayed by the ability of NRK-49F cells to form colonies in soft agar, by the ability to compete for binding to TGF-beta receptors on endothelial cells, and by an enzyme-linked immunosorbent assay selective for the active form of TGF-beta. Activation of latent TGF-beta by TSP stripped of associated TGF-beta activity (sTSP) is time- and concentration-dependent, but temperature-independent. The mechanism whereby sTSP activates latent TGF-beta appears to involve the direct binding of sTSP to the latent molecule as shown by gel permeation chromatography. In addition, a polyclonal antibody specific for the amino-terminal region of the latency-associated peptide (amino acids 81-94) inhibits sTSP-mediated activation of latent TGF-beta in both the chemically defined system and in endothelial cell conditioned medium. These data and the observation that similar concentrations of sTSP activate latent TGF-beta in both the chemically defined system and in the endothelial cell system indicate that there is a common mechanism by which TSP activates the small, large, and endothelial cell-derived latent TGF-beta complexes. The ability of TSP to convert latent TGF-beta to biologically active TGF-beta suggests that TSP is a major regulatory factor in the control of TGF-beta activity.

• Barcellos-Hoff MH, Derynck R, Tsang ML, Weatherbee JA
• Transforming growth factor-beta activation in irradiated murine mammary gland.
• J Clin Invest. 1994; 93: 892-9
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• The biological activity of TGF-beta, an important modulator of cell proliferation and extracellular matrix formation, is governed by dissociation of mature TGF-beta from an inactive, latent TGF-beta complex in a process that is critical to its role in vivo. So far, it has not been possible to monitor activation in vivo since conventional immunohistochemical detection does not accurately discriminate latent versus active TGF-beta, nor have events associated with activation been defined well enough to serve as in situ markers of this process. We describe here a modified immunodetection method using differential antibody staining that allows the specific detection of active versus latent TGF-beta. Under these conditions, we report that an antibody raised to latency-associated peptide detects latent TGF-beta, and we demonstrate that LC(1-30) antibodies specifically recognize active TGF-beta 1 in tumor xenografts overproducing active TGF-beta 1, without cross-reactivity in tumors expressing similar levels of latent TGF-beta 1. We previously reported that TGF-beta immunoreactivity increases in murine mammary gland after whole-body 60Co-gamma radiation exposure. Using differential antibody staining we now show that radiation exposure specifically generates active TGF-beta 1. While latent TGF-beta 1 was widely distributed in unirradiated tissue, active TGF-beta 1 distribution was restricted. Active TGF-beta 1 increased significantly within 1 h of irradiation concomitant with decreased latent TGF-beta immunoreactivity. This rapid shift in immunoreactivity provides the first evidence for activation of TGF-beta in situ. This reciprocal pattern of expression persisted for 3 d and was accompanied by decreased recovery of latent TGF-beta 1 from irradiated tissue. Radiation-induced activation of TGF-beta may have profound implications for understanding tissue effects caused by radiation therapy.

• Boyan BD et al.
• Latent transforming growth factor-beta is produced by chondrocytes and activated by extracellular matrix vesicles upon exposure to 1,25-(OH)2D3.
• J Biol Chem. 1994; 269: 28374-81
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• Resting zone and growth zone (GC) costochondral chondrocytes constitutively release latent, but not active, transforming growth factor-beta (TGF-beta) into the culture medium. When exogenous TGF-beta is added to the culture medium, no autocrine effect is observed. However, when 1,25-(OH)2D3 is added, a dose-dependent inhibition of latent TGF-beta release is found. Messenger RNA levels for TGF-beta 1 are unchanged by treatment with either 1,25-(OH)2D3 or TGF-beta 1. Since active growth factor was not observed in the conditioned medium, we tested the hypothesis that latent TGF-beta could be activated in the matrix. GC matrix vesicles, extracellular organelles associated with matrix calcification, were able to activate latent TGF-beta 1 and TGF-beta 2 when preincubated with 1,25-(OH)2D3. In contrast, GC plasma membranes activated latent TGF-beta, and addition of 1,25-(OH)2D3 inhibited this activation. The 1,25-(OH)2D3-dependent decrease in latent TGF-beta in the medium, with no detectable change in mRNA level, and the inhibition of plasma membrane activation of latent TGF-beta by 1,25-(OH)2D3 suggest that 1,25-(OH)2D3 may act through post-transcriptional and/or nongenomic mechanisms. The results also suggest that latent TGF-beta is activated in the matrix and that 1,25-(OH)2D3 regulates this activation by a direct, nongenomic action on the matrix vesicle membrane.

• Fukushima D, Butzow R, Hildebrand A, Ruoslahti E
• Localization of transforming growth factor beta binding site in betaglycan. Comparison with small extracellular matrix proteoglycans.
• J Biol Chem. 1993; 268: 22710-5
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• The most abundant binding molecule for transforming growth factor beta (TGF-beta) on many cell types is betaglycan, a transmembrane proteoglycan. To localize the binding site for TGF-beta in betaglycan, parts of the betaglycan extracellular portion were expressed as recombinant fusion proteins in bacteria and tested for their ability to compete for the binding of TGF-beta 1 to Hep G2 cells. One fragment encompassing 226 residues near the transmembrane domain (amino acids 543-769) (Lopez-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L., Lane, W. S., and Massague, J. (1991) Cell 67, 785-795) was active, whereas fusion proteins representing the other parts of the betaglycan ectodomain were inactive. Affinity measurements revealed two classes of binding sites (Kd = 3.9 nM and Kd = 145 nM) for the active fusion protein. The binding of the betaglycan fusion protein to immobilized TGF-beta 1 was inhibited by fusion proteins representing the core proteins of the small interstitial proteoglycans decorin, biglycan and fibromodulin, each also known to bind TGF-beta. The effective concentrations of TGF-beta for binding to these other proteoglycans were similar to those required for binding to betaglycan, indicating similar affinities for the binding of proteoglycans and betaglycan. Affinity cross-linking showed that, at low concentrations, the betaglycan fragment enhanced the binding of TGF-beta to the type II receptor and to endogenous betaglycan but had no effect on the binding to type I receptor. At high concentrations, the TGF-beta binding fragment inhibited the binding of TGF-beta to all these receptors. The fragment enhanced the activity of TGF-beta in mink lung cell bioassay at all active concentrations. The results indicate that betaglycan and the decorin type proteoglycans all bind to the same or closely spaced sites in TGF-beta and compete with one another for the binding. In addition, betaglycan may cooperate with the type II receptor on TGF-beta binding.

• Flaumenhaft R et al.
• Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells.
• J Cell Biol. 1993; 120: 995-1002
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• Transforming growth factor beta (TGF-beta) is released from cells in a latent form consisting of the mature growth factor associated with an aminoterminal propeptide and latent TGF-beta binding protein (LTBP). The endogenous activation of latent TGF-beta has been described in co-cultures of endothelial and smooth muscle cells. However, the mechanism of this activation remains unknown. Antibodies to native platelet LTBP and to a peptide fragment of LTBP inhibit in a dose-dependent manner the activation of latent TGF-beta normally observed when endothelial cells are cocultured with smooth muscle cells. Inhibition of latent TGF-beta activation was also observed when cells were co-cultured in the presence of an excess of free LTBP. These data represent the first demonstration of a function for the LTBP in the extracellular regulation of TGF-beta activity and indicate that LTBP participates in the activation of latent TGF-beta, perhaps by concentrating the latent growth factor on the cell surface where activation occurs.

• Tolsma SS, Volpert OV, Good DJ, Frazier WA, Polverini PJ, Bouck N
• Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.
• J Cell Biol. 1993; 122: 497-511
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• Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [3H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF beta 1, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to further localize inhibitory activity to two domains of the central stalk, the procollagen homology region and the properdin-like type 1 repeats. Trimeric recombinant TSP1 containing NH2-terminal sequences truncated after the procollagen-like module inhibited endothelial cell migration in vitro and corneal neovascularization in vivo whereas trimeric molecules truncated before this domain were inactive as was the NH2-terminal heparin-binding domain that is present in both recombinant molecules. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesis in vivo in the rat cornea and the migration of endothelial cells in vitro. A 19-residue peptide containing these sequences blocked vessel formation in the granulation tissue invading a polyvinyl sponge implanted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neovascularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 microM. One of these peptides, containing residues 481-499 of TSP1, also inhibited vessel formation in granulation tissue invading sponges in vivo. These results suggest that the large TSP1 molecule employs at least two different structural domains and perhaps two different mechanisms to accomplish a single physiological function, the inhibition of neovascularization. The definition of short peptides from each of these domains that are able to block the angiogenic process may be of use in designing targeted inhibitors of the pathological neovascularization that underlies many diseases.

• Colosetti P, Hellman U, Heldin CH, Miyazono K
• Ca2+ binding of latent transforming growth factor-beta 1 binding protein.
• FEBS Lett. 1993; 320: 140-4
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• Latent transforming growth factor-beta 1 binding protein (LTBP) is a constituent of the latent high molecular weight complex of TGF-beta 1 in human platelets. In the present communication, we show that LTBP binds Ca2+ in its free form as well as in the latent TGF-beta 1 complex. The binding of Ca2+ induces a structural change which protects the molecule against proteolysis and changes its elution position when analyzed by anion exchange chromatography. The in vitro activation of TGF-beta 1 is not influenced by the presence of Ca2+. The possible significance of Ca2+ binding of LTBP is discussed.

• Schultz-Cherry S, Murphy-Ullrich JE
• Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism.
• J Cell Biol. 1993; 122: 923-32
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• Thrombospondin (TSP) forms specific complexes with transforming growth factor-beta (TGF-beta) in the alpha granule releasate of platelets and these TSP-TGF-beta complexes inhibit the growth of bovine aortic endothelial cells (BAE). In these studies, we report that TSP stripped of associated TGF-beta (sTSP) retained growth inhibitory activity which was partially reversed by a neutralizing antibody specific for TGF-beta. Since BAE cells secrete latent TGF-beta, we determined whether sTSP activates the latent TGF-beta secreted by BAE cells. Cells were cultured with or without sTSP and then the conditioned medium was tested for the ability to support TGF-beta-dependent normal rat kidney (NRK) colony formation in soft agar. Medium conditioned with sTSP showed a dose- and time-dependent ability to stimulate BAE-secreted TGF-beta activity, reaching maximal activation by 1-2 h with 0.4 micrograms/ml (0.9 nM) sTSP. The sTSP-mediated stimulation of TGF-beta activity is not dependent on serum factors and is not a general property of extracellular matrix molecules. The sTSP-mediated stimulation of TGF-beta activity was blocked by a mAb specific for sTSP and by neutralizing antibodies to TGF-beta. Activation of BAE cell secreted latent TGF-beta by sTSP can occur in the absence of cells and apparently does not require interactions with cell surface molecules, since in conditioned medium removed from cells and then incubated with sTSP, activation occurs with kinetics and at levels similar to what is seen when sTSP is incubated in the presence of cells. Serine proteases such as plasmin are not involved in sTSP-mediated activation of TGF-beta. Factors that regulate the conversion of latent to active TGF-beta are keys to controlling TGF-beta activity. These data suggest that TSP is a potent physiologic regulator of TGF-beta activation.

• Okada F, Yamaguchi K, Ichihara A, Nakamura T
• Purification and structural analysis of a latent form of transforming growth factor-beta from rat platelets.
• J Biochem (Tokyo). 1989; 106: 304-10
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• A latent form of transforming growth factor type-beta (TGF-beta) with a high molecular weight was purified to homogeneity from rat platelets by a six-step procedure. The yield of the purified latent TGF-beta from platelets of 2,500 rats was 1.4 mg. The purified latent TGF-beta was activated by treatment with urea at concentrations of over 4M or acidic solutions of below pH 4. SDS-PAGE and gel filtration chromatography showed that the latent TGF-beta consisted of active TGF-beta and glycoproteins of about 200 kDa as masking components, and that under physiological conditions, these components formed a high molecular weight complex of about 400 kDa linked by non-covalent bonds. Here, we found that the masking protein was composed of one large subunit of about 110 kDa and two small subunits of 39 kDa linked by disulfide bridges. The N-terminal amino acid sequence of the small subunit was identical to the N-terminal region of the TGF-beta precursor lacking a signal peptide. From these findings, we proposed a structural model for the latent TGF-beta from rat platelets.

• Wakefield LM, Smith DM, Flanders KC, Sporn MB
• Latent transforming growth factor-beta from human platelets. A high molecular weight complex containing precursor sequences.
• J Biol Chem. 1988; 263: 7646-54
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• Human platelets, when induced to degranulate by thrombin, secrete transforming growth factor-beta (TGF-beta) in a biologically latent form. In this form, TGF-beta cannot bind to its cellular receptor, nor can it be immunoprecipitated by polyclonal antisera to TGF-beta, suggesting that the receptor-binding site and other TGF-beta epitopes may be masked. Western blot analysis of the platelet secretate indicates that the latent form of TGF-beta is a 220-235 kDa complex, in which mature TGF-beta (25 kDa) is noncovalently associated with sequences from the remainder of the precursor (74 kDa), and a third unidentified entity (approximately 135 kDa). The third component is immunologically unrelated to other growth factor binding proteins. The complex is glycosylated, and gel filtration analysis suggests it may exist in solution as higher molecular weight aggregates. Further chromatographic analysis indicates that in its latent form, the platelet TGF-beta cannot bind to alpha 2-macroglobulin (alpha 2M), but that if the platelet latent TGF-beta is activated by transient acidification, the released active TGF-beta will bind to alpha 2M. We have previously identified the latent form of TGF-beta found in serum as an alpha 2M.TGF-beta complex (O'Connor-McCourt, M. D., and Wakefield, L. M. (1987) J. Biol. Chem. 262, 14090-14099). We now propose that the latent TGF-beta secreted by platelets may be a cellular delivery complex, whereas the latent form found in serum may represent a clearance complex. Thus alpha 2M may scavenge excess TGF-beta that is released when the platelet latent form is activated, possibly by the clotting process. Finally, we have shown that the latent form of TGF-beta secreted by a variety of cell types in culture is similar, if not identical to that secreted by platelets.

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