Mast cell basic fibroblast growth factor in silicosis

To investigate the role of mast cells (MC) and their fibrogenic growth factors in silicosis, we performed quantitative immunohistochemistry for MC tryptase and for basic fibroblast growth factor (bFGF) in lung tissue from silicotic and control subjects. Anti-bFGF antibody was bound to lung MC, basement membrane, endothelial cells, and smooth-muscle cells. Morphometric analysis revealed that the volume density (V(v)) of MC was increased in silicotic lung and that the V(v) of bFGF-positive (bFGF(+)) cells was significantly higher than normal in silicotic lung. Most MC contained bFGF (rho = 0.88, p < 0.001). The V(v) of collagen/reticulin fibers was increased in silicosis and correlated with the V(v) of bFGF(+) cells (rho = 0.81, p < 0.001). Immature silicotic nodules contained bFGF(+) MC throughout the loose array of collagen/reticulin fibers. In large, mature nodules, the density of collagen/reticulin fibers was higher, and bFGF(+) MC were found only in the nodule periphery. Because of this circumferential MC alignment in silicotic nodules, we observed a negative correlation between the V(v) of bFGF(+) MC and the density of collagen/reticulin fibers in silicotic nodules (rho = -0.80, p < 0.001) and between the V(v) of all other nodule-associated cells and the density of collagen/reticulin fibers in the hypocellular nodule centers (rho = -0.84, p < 0.001). We conclude that MC that produce bFGF may play an important role in the development of silicosis.     

Insulin-like growth factor II stimulates cell proliferation through the insulin receptor

R− cells are 3T3-like fibroblasts generated from mouse embryos nullizygous for a targeted disruption of the genes encoding the type 1 insulin-like growth factor (IGF) receptor (IGF1R). These cells fail to proliferate in serum-free medium supplemented with purified growth factors, in contrast to their wild-type counterparts. However, when R− cells overexpress the insulin receptor from a stably integrated plasmid, R−/IR cells, they become capable of growing in serum-free medium supplemented solely with insulin or IGF-II, but not with IGF-I. Moreover, the introduction into R−/IR cells of an additional plasmid expressing IGF-II causes these cells to proliferate in serum-free medium without growth factor supplementation. From these results, we conclude that IGF-II can stimulate cell proliferation not only through its cognate IGF1R but also through the insulin receptor.     

Growth-associated protein GAP-43 and L1 act synergistically to promote regenerative growth of Purkinje cell axons in vivo

Neuronal expression of growth-associated protein 43 (GAP-43) and the cell adhesion molecule L1 has been correlated with CNS axonal growth and regeneration, but it is not known whether expression of these molecules is necessary for axonal regeneration to occur. We have taken advantage of the fact that Purkinje cells do not express GAP-43 or L1 in adult mammals or regenerate axons into peripheral nerve grafts to test the importance of these molecules for axonal regeneration in vivo. Transgenic mice were generated in which Purkinje cells constitutively express L1 or both L1 and GAP-43 under the Purkinje cell-specific L7 promoter, and regeneration of Purkinje cell axons into peripheral nerve grafts implanted into the cerebellum was examined. Purkinje cells expressing GAP-43 or L1 showed minor enhancement of axonal sprouting. Purkinje cells expressing both GAP-43 and L1 showed more extensive axonal sprouting and axonal growth into the proximal portion of the graft. When a predegenerated nerve graft was implanted into double-transgenic mice, penetration of the graft by Purkinje cell axonal sprouts was strongly enhanced, and some axons grew along the entire intracerebral length of the graft (2.5-3.0 mm) and persisted for several months. The results demonstrate that GAP-43 and L1 coexpressed in Purkinje cells can act synergistically to switch these regeneration-incompetent CNS neurons into a regeneration-competent phenotype and show that coexpression of these molecules is a key regulator of the regenerative ability of intrinsic CNS neurons in vivo.     

Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor.

A human hepatoma cell line synthesizes, as evidenced by metabolic labeling, an endothelial cell mitogen that is found to be mostly cell associated. The hepatoma-derived growth factor (HDGF) has been purified to homogeneity by a combination of Bio-Rex 70, heparin-Sepharose, and reverse-phase chromatography; it is a cationic polypeptide with a molecular weight of about 18,500-19,000. HDGF is structurally related to basic fibroblast growth factor (FGF). Immunological analysis demonstrates that antiserum prepared against a synthetic peptide corresponding to the amino-terminal sequence of basic FGF cross-reacts with HDGF when analyzed by electrophoretic blotting and by immunoprecipitation. Sequence analysis of tryptic fragments demonstrates that HDGF contains sequences that are homologous to both amino-terminal and carboxyl-terminal sequences of basic FGF.     

Insulin-like growth factor II stimulates motor nerve regeneration.

Injury to mammalian motor nerves can lead to paralysis, but relatively successful regeneration may occur when conditions are favorable. Elucidation of the mechanism upholding successful regeneration is of theoretical and clinical interest. In this study, the hypothesis that insulin-like growth factor II (IGF-II) can stimulate motor nerve regeneration was tested. When IGF-II was infused continuously near a site of crush on the sciatic nerve, the distance of motor axon regeneration was increased significantly in rats. In contrast, spontaneous regeneration was inhibited when an anti-IGF-II antiserum was infused through a "window" in the epineurium. Thus, infused IGF-II can increase, and endogenous IGFs can support, the regeneration of motor axons in lesioned nerves.     

Stem cell factor and basic fibroblast growth factor are synergistic in augmenting committed myeloid progenitor cell growth

Stem cell factor (SCF) and basic fibroblast growth factor (bFGF) are hematopoietic cytokines produced by bone marrow stromal cells. It is known that, although SCF and bFGF have limited clonogenic activity on their own, they can augment colony-stimulating factor (CSF)-mediated progenitor cell growth. Because these factors are both sequestered by stromal cells, we examined their interaction on progenitor cell growth in conjunction with granulocyte-macrophage-CSF (GM-CSF). In this study, we show that clonogenic growth derived from low-density bone marrow cells stimulated by GM-CSF is significantly augmented (P < .001) in the presence of maximal (100 ng/mL) concentrations of SCF in combination with 100 ng/mL of bFGF. When CD34+ cells are used, the synergistic effect of bFGF and SCF for GM-CSF-mediated progenitor cell growth is further increased, resulting in as much as a sevenfold increase in detectable colony-forming units granulocyte-macrophage (P < .001). These data suggest that the synergistic activity of bFGF and SCF is mediated directly on hematopoietic precursors. These observations suggest that bFGF and SCF, concentrated locally on stromal cell surfaces, might interact in concert with other hematopoietic cytokines to regulate stem cell proliferation and differentiation in hematopoietic niches in the bone marrow.     

Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor beta

We have tested the hypothesis that basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) regulate the proliferation of osteoblast-like cells. Cells which migrated from central bone explants of fetal calf calvaria expressed markers characteristic of the osteoblast phenotype, including osteocalcin (bone Gla protein) secretion and increased cAMP production in response to treatment with PTH. Bone cells proliferated in response to bFGF in a dose- and time-dependent pattern (ED50 = 60 pg/ml media). bFGF increased both the rate of bone cell proliferation (1.7-fold above controls) and final cell density at confluence (3-fold above controls). Acidic FGF (aFGF) exerted comparable effects though with lesser potency (ED50 = 2 ng/ml). In addition to its mitogenic effect, bFGF increased the osteocalcin content of conditioned media, suggesting that bFGF also modulates the function of osteoblast-like cells. Although TGF beta did not stimulate bone cell proliferation, it potentiated the mitogenic effects of aFGF and bFGF. In the presence of bFGF (0.7 ng/ml) the response to TGF beta was dose-dependent (ED50 = 1.7 ng/ml), with maximal stimulation at 5 ng/ml. These results demonstrate that aFGF and bFGF are mitogenic for bone cells in vitro. Furthermore, TGF beta potentiates the effects of bFGF and aFGF on the proliferation of bone cells. Since these growth factors are present in bone tissue in vivo, these data support the proposal that FGF and TGF beta may participate in the regulation of bone formation.     

Three-dimensional structure of human basic fibroblast growth factor.

The three-dimensional structure of human basic fibroblast growth factor (bFGF) has been determined by x-ray crystallography and refined to a crystallographic residual of 17.4% at 2.2-A resolution. The structure was initially solved at a nominal resolution of 2.8 A by multiple isomorphous replacement using three heavy-atom derivatives. Although the map clearly showed the overall fold of the molecule, electron density was not observed for the first 19 amino-terminal and the last 3 carboxyl-terminal amino acids, suggesting that they are disordered. The bFGF crystals were grown from 2.0 M ammonium sulfate at pH 8.1 in space group P1 with cell dimensions a = 30.9 A, b = 33.4 A, c = 35.9 A, alpha = 59.5 degrees, beta = 72.0 degrees, and gamma = 75.6 degrees. There is one molecule per unit cell and the crystals diffract to spacings beyond 1.9 A. The overall structure of bFGF can be described as a trigonal pyramid with a fold very similar to that reported for interleukin 1 beta, interleukin 1 alpha, and soybean trypsin inhibitor. An apparent sulfate ion is bound within a basic region on the surface of the molecule and has a ligands the main-chain amide of Arg-120 and the side chains of Asn-27, Arg-120, and Lys-125. This is suggested as the presumed binding site for heparin. Residues 106-115, which are presumed to bind to the bFGF receptor [Baird, A., Schubert, D., Ling, N. & Guillemin, R. (1988) Proc. Natl. Acad. Sci. USA 85, 2324-2328], include an irregular loop that extends somewhat from the surface of the protein and is about 25 A from the presumed heparin binding site. The backbone structure of this putative receptor-binding loop is very similar, although not identical, to the corresponding region of interleukin 1 beta.     

Pituitary follicular cells produce basic fibroblast growth factor.

Cultured monolayers of bovine pituitary follicular cells, which transport ions, contain high amounts of mitogenic activity for endothelial cells which, on the basis of gene expression analysis, heparin-Sepharose elution profile, bioassay, immunoblotting, radioimmunoassay, and radioreceptor assay, has been identified as basic fibroblast growth factor (bFGF). These data indicate that follicular cells may be a major source of bFGF in the pituitary gland. Considering that bFGF has been proposed to play a role in paracrine regulation of pituitary hormone secretion, the data also suggest that these cells may exert important local regulatory functions.     

Receptor- and heparin-binding domains of basic fibroblast growth factor.

Two functional domains in the primary structure of basic fibroblast growth factor (FGF) have been identified on the basis of their ability to interact with the FGF receptor, bind radiolabeled heparin, and modulate the cellular response to FGF. Peptides derived from these two functional domains can act as partial agonists and antagonists in biological assays of FGF activity. Peptides related to the sequences of FGF-(24-68)-NH2 and FGF-(106-115)-NH2 inhibit thymidine incorporation into 3T3 fibroblasts when they are stimulated by FGF but have no effect when the cells are treated with either platelet-derived growth factor or epidermal growth factor. They also possess partial agonist activity and can stimulate DNA synthesis when tested in the absence of exogenous FGF. The active peptides have no effect on the binding of epidermal growth factor to its receptor on A431 cells and they can modulate the effects of FGF, but not fibronectin, on endothelial cell adhesion. The results suggest the possibility of designing specific analogs of FGF that are capable of inhibiting the biological effects of FGF.     

Insulin-like growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor

Summary The release of growth factors from ischaemic retina has been hypothesized as the central stimulus for retinal neovascularization in proliferative diabetic retinopathy. Two of the growth factors implicated are insulin-like growth factor-I and basic fibroblast growth factor. We examined the effect of insulin-like growth factor-I on in vivo neovascularization using the established angiogenic model of the rabbit cornea (n=30), and also compared the effects of insulin-like growth factor-I and basic fibroblast growth factor using two new in vivo systems. Either supraphysiologic concentrations of each growth factor (600 g) were injected intravitreally into pigmented rabbits (n=21) or porous polyfluorotetraethylene chambers filled with an emulsion containing collagen and growth factor (500 ng) were placed on the retina surface (n=8). Our results demonstrate that when insulin-like growth factor-I was implanted together with a slow release carrier into the pocket of the normally avascular cornea, insulin-like growth factor-I (10 g/pellet) induced angiogenesis in all rabbits. This degree of angiogenesis was comparable to that previously shown for basic fibroblast growth factor. For the intravitreal studies, the fibrotic component was greater in the basic fibroblast growth factor injected eyes, whereas the vascular component was accentuated in the eyes injected with insulin-like growth factor-I. Light and electron microscopy demonstrated areas of vascular proliferation in both groups. Porous polyfluorotetraethylene chamber studies with insulin-like growth factor-I and basic fibroblast growth factor demonstrated vascular proliferation in the vicinity of the chamber similar to the intravitreal injected eyes, but to a lesser degree than the injected eyes. Our experiments overall support the angiogenic potential of both insulin-like growth factor-I and basic fibroblast growth factor and support distinct but complimentary roles for each growth factor in the pathogenesis of proliferative diabetic retinopathy.     

High-affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding.

We have isolated RNA ligands with low-nanomolar affinity and high specificity to basic fibroblast growth factor from a pool of 10(14) molecules containing 30 randomized positions by the systematic evolution of ligands by exponential enrichment (SELEX) procedure. High-affinity ligands could be classified into two families based on sequence and secondary structure similarities. Representative RNA ligands from the two families compete with one another as well as with heparin for binding to the protein. Furthermore, we show that these ligands inhibit the first step in the signaling pathway of basic fibroblast growth factor: binding of the growth factor to its cell-surface receptors. These findings emphasize the general usefulness of SELEX as a tool for discovering potent, specific oligonucleotide antagonists of target proteins.     

Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), which stimulate the phosphorylation of proteins on tyrosine in PC12 cells, initiate these modifications through ligand-specific cell surface receptors that contain the causative tyrosine kinases. One apparent substrate for these enzymes is phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates the D-3 position of the inositol ring and associates with several protein tyrosine kinases, as indicated by the fact that it is immunoprecipitated from EGF-, bFGF-, and NGF-stimulated PC12 cells by an anti-phosphotyrosine antibody. All three growth factors increase immunoprecipitable PI 3-kinase activity after 2 min of addition at concentrations able to stimulate either mitogenic or neurotrophic responses in PC12 cells. The level of stimulation of PI 3-kinase activity by EGF, bFGF, and NGF is 15- to 20-fold, 2- to 3-fold, and 8- to 10-fold, respectively. Moreover, tyrosine phosphorylation of PI 3-kinase was detected in EGF-, bFGF-, and NGF-stimulated PC12 cells, and the amount of the phosphorylation correlated with the level of stimulation of enzyme activity. In contrast, phosphatidylinositol 4-kinase, which produces the inositol phospholipids cleaved by phospholipase C-gamma to yield diacylglycerol and inositol-1,4,5-trisphosphate, is not affected by these growth factors. The pattern of stimulation of PI 3-kinase does not correlate with the induction of neurite outgrowth but rather with the mitotic responses, suggesting that PI 3-kinase and its products may be more important for signaling in cell division than in trophic processes. However, the levels of phosphatidylinositol 3-phosphate do not coincide with the stimulation of [3H]thymidine incorporation by these growth factors, rendering its role in mitotic functions, at least in PC12 cells, also uncertain.     

Early changes in protein synthesis induced by basic fibroblast growth factor, nerve growth factor, and epidermal growth factor in PC12 pheochromocytoma cells.

Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate neuronal differentiation, whereas epidermal growth factor (EGF) promotes only mitogenic responses in PC12 pheochromocytoma cells. The early changes in protein synthesis induced by bFGF, NGF, and EGF in these cells have been determined by two-dimensional PAGE of [35S]methionine-labeled proteins and computerized image analysis. The rate of synthesis of only 29 proteins (out of approximately 1500 identified) was found to be modulated during the first several hours of growth factor stimulation. Individually, 12 were affected by EGF, 23 were affected by bFGF, and 20 were affected by NGF. Eight of these were regulated by all three growth factors, while 10 proteins were commonly induced by bFGF and NGF, in accordance with the essentially identical morphological responses induced by these two factors. In addition, the effects of bFGF and NGF were about equally divided between increases and decreases in the rate of synthesis of individual proteins, whereas EGF caused significantly more positive (increased) responses. All proteins modulated by NGF or FGF alone were negative in their response and those induced by only EGF were positive. Of particular interest, the rate of synthesis of two proteins of 55 kDa and pI 5.45 and 5.50 was dramatically and transiently induced during the first 2 hr of bFGF and NGF treatment and was not affected by EGF. This study indicates that all three factors elicit early increases and decreases in the synthesis of a quite limited number of proteins and provides molecular evidence for the specificity of a differentiative vs. a proliferative growth factor-induced signaling pathway in these cells.     

Transforming growth factor beta and follicle-stimulating hormone promote rat granulosa cell proliferation

Rat granulosa cells isolated from the ovaries of diethylstilbestrol-primed immature rats were treated with estrogen, FSH, and growth factors to determine those factors that were required to promote DNA synthesis. Estrogen and FSH, previously shown to stimulate the incorporation of [3H]thymidine into rat granulosa cell DNA in vivo, were ineffective in vitro. Epidermal growth factor, insulin-like growth factor 1 (IGF1), and fibroblast growth factor did not influence DNA synthesis whereas transforming growth factor beta (TGF beta) alone had a significant effect. Neither estradiol-17 beta (5 X 10(-8)-5 X 10(-6) M) nor IGF1 augmented the actions of TGF beta and FSH. FSH did not influence the actions of epidermal growth factor or IGF1 but dramatically augmented the effect of TGF beta on DNA synthesis. FSH and TGF beta also stimulated [3H]thymidine incorporation into the DNA of granulosa cells isolated from immature rats not treated with diethylstilbestrol. The increase in [3H]thymidine incorporation into DNA stimulated by TGF beta and FSH resulted subsequently in an increase in cell number. The response of the cells to TGF beta in the presence of a constant level of FSH (10 ng/ml) was dose dependent, 2.5 ng/ml being the minimal effective concentration. In the presence of antibody specific for TGF beta the bioactivity of the TGF beta was neutralized indicating that the growth promoting activity was due to TGF beta and not due to contaminants. In this paper, we have shown that the combined actions of FSH and TGF beta influence DNA synthesis and the proliferation of rat granulosa cells. Interactions between FSH and TGF beta may be important in regulating aspects of rat granulosa cell growth in addition to exerting pronounced effects on cytodifferentiation.     

Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor.

Primary functional bovine adrenal cortical cell cultures have been developed to study the factors controlling adrenal cell growth. Cells were prepared by the collagenase technique and maintained in F-12 medium containing fetal calf serum and horse serum. Cells contained abundant lipid as demonstrated by staining with Oil Red O and showed strongly positive staining for delta5,3beta-hydroxysteroid dehydrogenase. ACTH inhibited DNA synthesis and stimulated steriodogenesis in these cells. Fibroblast growth factor (FGF) was shown to be a potent stimulator of the growth of normal bovine adrenal cortical cells maintained in tissue culture. The minimal effective dose of FGF was 1 ng/ml with maximal effects being observed at 100 ng/ml. The effect of FGF was dependent on the serum concentration. Inclusion of FGF in F-12 medium containing serum permitted cloning of functional bovine adrenal cortical cells from cultures seeded at low density (4 cells/cm2). ACTH inhibited the mitogenic effects of FGF. In addition to its mitogenic action, FGF is a migratory factor for bovine adrenal cortical cells. Though ACTH inhibited the mitogenic effects of FGF, it did not block the migratory activity. Epidermal growth factor did not affect the growth of either normal bovine adrenal or functional mouse adrenal tumor cells (Y-1) in tissue culture. FGF is the first direct mitogen identified for adrenal cortical cells; ACTH opposes this mitogenic action and functions directly as a differentiate function signal.