Transforming growth factor activity of bovine brain-derived growth factor

Bovine brain-derived growth factor (BDGF), whose biochemical properties resemble those of endothelial cell growth factor (ECGF) and brain-derived acidic fibroblast growth factor (acidic FGF), is able to promote colony formation of normal rat kidney fibroblasts (NRK cells) in soft agar. As in the case of transforming growth factor beta (TGF beta), EGF potentiates the anchorage-independent growth promoting activity of BDGF. In the presence of EGF (5 ng/ml), the optimal concentration of BDGF for stimulation of anchorage-independent of NRK cells is approximately 0.5 ng/ml. At higher concentrations, BDGF becomes inhibitory. The anchorage-independent cell growth promoting activity of BDGF differs from that of TGF beta in acid and reducing agent stability.     

Transforming growth factor beta 1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane

Application of TGF beta 1 (10-100 ng) to the chicken chorioallantoic membrane (CAM) for 72 h resulted in a dose-dependent, gross angiogenic response. The vascular effects induced by TGF beta 1 were qualitatively different than those induced by maximal doses of basic FGF (bFGF) (500 ng). While TGF beta 1 induced the formation of large blood vessels by 72 h, bFGF induced primarily small blood vessels. Histologic analysis revealed that TGF beta 1 stimulated pleiotropic cellular responses in the CAM. Increases in fibroblast and epithelial cell density in the area of TGF beta 1 delivery were observed as early as 4 h after TGF beta 1 treatment. By 8 h, these cell types also demonstrated altered morphology and marked inhibition of proliferation as evidenced by 3H-thymidine labeling. Thus, the TGF beta 1-stimulated accumulation of these cell types was the result of cellular chemotaxis from peripheral areas into the area of TGF beta 1 delivery. Microscopic angiogenesis in the form of capillary sprouts and increased endothelial cell density first became evident at 16 h. By 24 h, capillary cords appeared within the mesenchyme of the CAM, extending towards the point of TGF beta 1 delivery. 3H-thymidine labeling revealed that the growth of these capillary cords was due to endothelial cell proliferation. Finally, perivascular mononuclear inflammation did not become evident until 48 h of treatment, and its presence correlated spatially and temporally with the gross and histological remodelling of newly formed capillary cords into larger blood vessels. In summary, these data suggest that, in the chicken CAM, TGF beta 1 initiates a sequence of cellular responses that results in growth inhibition, cellular accumulation through migration, and microvascular angiogenesis.     

Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells

Transforming growth factor beta-1 (TGF beta-1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGF beta-1, but it is blocked if cells are cultured on extracellular-matrix-coated dishes instead of plastic. TGF beta-1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGF beta-1 might act through regulation of bFGF synthesis in BCE cells.     

Epithelial-mesenchymal cell transformation in the embryonic heart can be mediated, in part, by transforming growth factor beta

Progenitor cells of the valves and membranous septa of the vertebrate heart are formed by transformation of a specific population of endothelial cells into mesenchyme. Previous studies have shown that this epithelial-mesenchymal cell transformation is mediated by a signal produced by the myocardium of the atrioventricular (AV) canal and transferred across the extracellular matrix. Data are presented here that transforming growth factor beta (TGF beta 1 or TGF beta 2), in combination with an explant of ventricular myocardium, will produce an epithelial-mesenchymal transformation by cultured AV canal endothelial cells in vitro. Alone, neither component is capable of producing this effect. The factor provided by the ventricular explant cannot be substituted by either epidermal growth factor or basic fibroblast growth factor. Further experiments show that an antibody that blocks TGF beta activity is effective in preventing the epithelial-mesenchymal cell transformation normally produced by AV canal myocardium. Control antibodies are without effect. By immunological criteria, a member of the TGF beta family of molecules can be demonstrated in the chicken embryo and heart at the time overt valvular formation begins. Together, these data show that TGF beta 1 can produce mesenchymal cell formation in vitro and provide evidence that a member of the TGF beta family is present and plays a role in the process of epithelial-mesenchymal cell transformation in the embryonic heart.     

Inhibition of myogenic differentiation by fibroblast growth factor or type beta transforming growth factor does not require persistent c-myc expression

Skeletal muscle differentiation is accompanied by accumulation of the mRNA encoding the muscle isoenzyme of creatine kinase (MCK) and can be suppressed by serum components, fibroblast growth factor (FGF), or type beta transforming growth factor (TGF beta). Using the nonfusing myogenic cell line, BC3H1, the potential involvement of c-myc in growth factor-dependent inhibition of myogenesis was examined. Withdrawal of undifferentiated myoblasts from the cell cycle in medium with 0.5% serum was associated with a precipitous decline in expression of c-myc mRNA followed by induction of MCK mRNA. In 0.5% serum containing TGF beta, c-myc mRNA declined to a level identical to that in differentiated cells; however, MCK mRNA was not expressed. Exposure of quiescent differentiated cells to FGF or TGF beta caused disappearance of muscle-specific gene products and was accompanied by only transient low level induction of c-myc mRNA. These data indicate that persistent c-myc expression is not required for growth factor-mediated inhibition of myogenic differentiation.     

Transforming growth factors and control of neoplastic cell growth

Transforming growth factors (TGFs) are peptides that affect the growth and phenotype of cultured cells and bring about in nonmalignant fibroblastic cells phenotypic properties that resemble those of malignant cells. Two types of TGFs have been well characterized. One of these, TGF alpha, is related to epidermal growth factor (EGF) and binds to the EGF receptor, whereas the other, TGF beta, is not structurally or functionally related to TGF alpha or EGF and mediates its effects via distinct receptors. TGF beta is produced by a variety of normal and malignant cells. Depending upon the assay system employed, TGF beta has both growth-inhibitory and growth-stimulating properties. Many of the mitogenic effects of TGF beta are probably an indirect result of the activation of certain growth factor genes in the target cell. The ubiquitous nature of the TGF beta receptor and the production of TGF beta in a latent form by most cultured cells suggests that the differing cellular responses to TGF beta are regulated either by events involved in the activation of the factor or by postreceptor mechanisms. The combined effects of TGF beta with other growth factors or inhibitors evidently play a central role in the control of normal and malignant cellular growth as well as in cell differentiation and morphogenesis. Since transforming growth factor as a concept has partially proven misleading and insufficient, there is a need to find a new nomenclature for these regulators of cellular growth and differentiation.     

A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.

An endothelial cell line (M40) resistant to growth inhibition by transforming growth factor-beta type 1 (TGF beta 1) was isolated by chemical mutagenesis and growth in the presence of TGF beta 1. Like normal endothelial cells, this mutant is characterized by high expression of type II TGF beta receptor and low expression of type I TGF beta receptor. However, the mutant cells display a type II TGF beta receptor of reduced molecular weight as a result of a general defect in N-glycosylation of proteins. The alteration does not impair TGF beta 1 binding to cell surface receptors or the ability of TGF beta 1 to induce fibronectin or plasminogen activator inhibitor-type I production. M40 cells were also resistant to growth inhibition by tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) but were inhibited by interferon-gamma (IFN gamma) and heparin. These results imply that TGF beta 1, TNF alpha, and IL-1 alpha act through signal transducing pathways that are separate from pathways for IFN gamma and heparin. Basic fibroblast growth factor was still mitogenic for M40, further suggesting that TGF beta 1, TNF alpha, and IL-1 alpha act by direct inhibition of cell growth rather than by interfering with growth stimulatory pathways.     

Comparison of the biological actions of TGF beta-1 and TGF beta-2: differential activity in endothelial cells

Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-1 and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-1 and TGF beta-2 inhibited cell proliferation in MvILu with no significant differences in the ED50 (31 +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-1 in inhibiting DNA synthesis in AEC (6310 +/- 985 pg/ml vs 101 +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-1 and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs 10 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF), skeletal growth factor (SGF)/insulin-like growth factor-II (IGF-II), and insulin-like growth factor-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta-1, both acidic and basic FGF significantly reduced TGF beta-1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-1, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-1 from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-1 and TGF beta-2, and (4) both TGF beta-1 and TGF beta-2 are potent mitogens for chicken embryonic calvarial bone cells.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis.

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGF beta), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGF beta and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGF beta to bFGF determines the magnitude of the Schwann cell response.     

Retina- and eye-derived endothelial cell growth factors: partial molecular characterization and identity with acidic and basic fibroblast growth factors

Two retina-derived growth factors have been isolated on the basis of their ability to stimulate the proliferation of capillary endothelial cells in vitro. Gas-phase sequence analysis identified the amino-terminal sequence of the major form of the mitogen as being identical with residues 1-35 of bovine basic fibroblast growth factor (FGF). Amino-terminal sequence analysis of the second form identified 28 residues that are indistinguishable from those of brain acidic FGF (residues 1-28). The possibility that these retina-derived endothelial cell growth factors are related to, if not identical with, basic and acidic FGF is supported by observations that they have similar molecular weights (15000-16000), similar retention behavior on all steps of chromatography (ion-exchange, heparin-Sepharose), and similar amino acid compositions and that they cross-react with antibodies to basic and acidic FGF. The eye-derived growth factors, like FGF, are potent stimulators of capillary endothelial cell growth in vitro. The results identify the major retina-derived endothelial cell growth factor as indistinguishable from basic FGF and demonstrate the presence of an acidic FGF in the eye. They suggest that at least some of the mitogenic, angiogenic, and neovascularizing activities described as being present in the retina are due to the existence of FGF in this tissue. The implications of this finding on the etiology and pathophysiology of vasoproliferative diseases of the eye are discussed.     

Peptide growth factor interactions in embryonic and fetal growth.

Peptide growth factors are expressed by multiple tissues in the animal and human embryo and fetus. They undergo specific interactions which control the rate of cellular proliferation, tissue differentiation and the induction of specific morphogenic events such as mesoderm formation in the embryo. Biologic control may not only be exerted at the level of growth factor synthesis and receptor expression but by the sequestration and storage of growth factors by extracellular matrix molecules. In the case of insulin-like growth factors (IGFs), storage maybe mediated by attachment to specific IGF-binding proteins which may additionally modulate biological potency. Basic fibroblast growth factor (basic FGF) and transforming growth factor-beta (TGF beta) directly bind to glycosaminoglycan molecules. Release of growth factors from these stores may be by local proteolytic action. A sequential expression of basic FGF, IGF-II and TGF beta occurs in the ovine fetal epiphyseal growth plate as chondrocytes progress from a proliferative to a postmitotic, hypertrophic state. Cellular phenotype may be largely explained by the relative amounts of these autocrine growth factors within the growth plate.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGFβ), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGFβ and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGFβ to bFGF determines the magnitude of the Schwann cell response.     

Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.     

Transforming growth factor beta 1-specific binding proteins on human vascular endothelial cells.

Transforming growth factor beta (TGF beta) regulates the growth of human umbilical vein endothelial cells (HUVEC) differently depending on the isoform of TGF beta and the culture conditions. The cells are resistant to growth inhibition by TGF beta when the cells are cultured on substratum coated with gelatin. However, the proliferation of HUVEC cultured on substratum without a gelatin coating is inhibited by TGF beta, depending on the isoform and concentration of TGF beta. Binding assays with 125I-TGF beta 1 reveal that HUVEC contain a single class of high-affinity (Kd = 4.4 pM) TGF beta 1 binding sites with 8500 sites per cell. Affinity cross-linking studies demonstrate that HUVEC express 180 and 80 kDa TGF beta 1 binding sites that do not bind TGF beta 2. The reduction and the removal of glycosaminoglycans does not affect the electrophoretic mobility of the 180-kDa binding protein cross-linked with 125I-TGF beta 1. Therefore, the 180-kDa TGF beta 1 binding protein is not related to the type III TGF beta receptor, but might be a novel TGF beta 1-specific receptor/binding protein expressed on vascular endothelial cells. The expression of TGF beta 1 binding sites is not affected by the presence or absence of the gelatin coating on the culture substratum. The data suggest that a gelatin coating does not regulate the susceptibility of HUVEC to TGF beta 1 at the level of the receptor/binding proteins, and that growth inhibition of HUVEC by TGF beta 1 is linked to the regulation of extracellular matrices required for the interaction between the cells and the substratum.     

Activation of p21ras by transforming growth factor beta in epithelial cells.

The transforming growth factor beta (TGF beta) family members are ubiquitously expressed and control a variety of cellular processes by interacting with at least two types of high affinity cell surface receptors. However, the primary signal transduction mechanism of the receptors is unknown. The ras-encoded 21-kDa GTP binding proteins have recently been shown to mediate the effects of other polypeptide growth factors. Here we show that both TGF beta 1 and TGF beta 2 (5 ng/ml) result in a rapid (within 6 or 12 min, respectively) stimulation of GTP bound to p21ras in TGF beta-sensitive intestinal epithelial cells. Further, the CCL64 epithelial cell line, extremely sensitive to growth inhibition by TGF beta, displayed a concentration-dependent increase in GTP bound to p21ras by TGF beta 1 and a rapid activation of p21ras by TGF beta 2. The results provide the first direct evidence for rapid activation of a receptor coupling component for TGF beta in epithelial cells.     

The effect of Pygeum africanum on fibroblast growth factor (FGF) and transforming growth factor beta (TGF beta 1/LAP) expression in animal model

On the basis of its fibroblast growth factor (FGF) inhibitory effect we assessed the possible inhibitory anti-inflammatory role of Pygeum Africanum extract (Tadenan) on FGF and transforming growth factor beta (TGF beta 1/LAP) expression of macrophages and neutrophils in broncho-alveolar lavage fluid (BAL) of rats in a bleomycin-induced acute inflammation model. The rats were divided into three groups: 17 untreated controls, 10 bleomycin-instilled rats, receiving NaCl (0.9%), and 10 rats receiving Pygeum Africanum extract. On the 12th (and 15th day) we performed BAL and after labelling of cells expression of FGF and TGF beta 1 (LAP) was measured by flow-cytometry. We made a quantitative analysis of BAL cells as well. One-way ANOVA was used for statistical analysis. We found in Pygeum Africanum extract treated group 1, a significantly decreased number of neutrophil granulocytes (p < 0.05) compared with other groups 2, there was a considerable decrease (not significant) in expression of TGF beta 1(LAP) on BAL macrophages, but not in case of FGF. In conclusion: our results show the possible 1. inhibitory effect of this drug on TGF beta 1 (LAP) expression, 2. anti-inflammatory role on neutrophil granulocytes.     

Transforming growth factor-beta 1 overproduction in prostate cancer: effects on growth in vivo and in vitro.

We found previously that transforming growth factor-beta 1 (TGF beta 1) mRNA levels are markedly elevated in rat prostate cancer (Dunning R3327 sublines) compared to levels in normal prostate. Our goal was to determine whether elevated expression of TGF beta 1 is biologically relevant to prostate cancer growth in vivo. We chose as our model the R3327-MATLyLu prostate cancer epithelial cell line, which produces metastatic anaplastic tumors when reinoculated in vivo. Our approach was to stably transfect MATLyLu cells with an expression vector that codes for latent TGF beta 1 and to isolate subclones of cells that over-expressed TGF beta 1 mRNA. We also isolated a subclone of MATLyLu cells transfected with a control vector lacking the TGF beta 1 cDNA insert. We then studied the growth of these cells in vivo and in vitro. Twenty days after sc inoculation of 10(6) cells in vivo, TGF beta 1-overproducing MATLyLu tumors were 50% larger, markedly less necrotic, and produced more extensive metastatic disease (lung metastases in 73% of all lobes and lymph node metastases in 88% of animals) compared to control MATLyLu tumors (lung metastases, 21%; lymph node metastases, 7%). Thus, TGF beta 1 produced in vivo is biologically active and can promote prostate cancer growth, viability, and aggressiveness, perhaps via effects on the host and/or on the tumor cells themselves. When followed in vitro, TGF beta 1-overproducing cells became growth inhibited, but this effect was transient as cells subsequently resumed proliferating. Growth inhibition was due to TGF beta, because it could be prevented by TGF beta-neutralizing antibody. Therefore, prostate cancer cells can activate and respond to secreted latent TGF beta 1, and although the cells are transiently inhibited in vitro, there is no net inhibition of growth. The ability of the cells to respond to endogenously produced TGF beta 1 suggests that TGF beta 1 overexpression enhances tumor growth in vivo at least in part via an effect of TGF beta 1 on the tumor cells themselves.     

Human transforming growth factor-beta 3: recombinant expression, purification, and biological activities in comparison with transforming growth factors-beta 1 and -beta 2

Recent cDNA characterization has predicted the existence of a new member of the transforming growth factor family, transforming growth factor-beta 3 (TGF beta 3). However, nothing is known about the biological activities of the TGF beta 3 protein, since it has not been purified from any natural sources. We report here the recombinant expression in mammalian cells and the purification to apparent homogeneity of human TGF beta 3. The TGF beta 3 was evaluated in comparison with purified TGF beta 1 and TGF beta 2 in several assays for its effects on stimulation or inhibition of proliferation of mammalian cells. These analyses revealed that TGF beta 3 exerts activities similar to the two other TGF beta species, but that there are distinct differences in potencies between the different TGF beta forms depending on the cell type and assay used.     

Transforming growth factor-β inhibits cellular adenylate cyclase activity in cultured human arterial endothelial cells

Summary Stimulation of human arterial endothelial cells with heparin-binding growth factor-1 (HBGF-1) resulted in a 40% to 60% increase in the cellular adenylate cyclase activity and intracellular cAMP content. The stimulatory effect of HBGF-1 was effectively suppressed by pretreating the cells with transforming growth factor-β (TGF-β), an endothelial cell growth inhibitor. The inhibition of the adenylate cyclase activity precedes growth inhibition by at least 24 h. The half maximal inhibitory dose was calculated to be 0.2 ng/ml for the inhibition of both cyclase activity and cell growth. The possible role of the adenylate cyclase suppression in growth inhibition by TGF-β is discussed. This work was supported in part by grants from NCI (CA 37589), RJR Nabisco, Inc. and Kyowa Hakko Kogyo, Co., Ltd. Editor's Statement The observation that heparin-binding growth factor activates adenylate cyclase in endothelial cells and TGF beta lowers cAMP levels in endothelial cells treated with heparin-binding growth factor raises the possibility that growth control may be mediated, at least partially, through cyclic nucleotides in this system, as well as raising questions about relationships between activities of these peptide growth factors and G protein activation.