Mutants of Basic Fibroblast Growth Factor Identify Different Cellular Response Programs

Abstract

Mutations, expected to affect the intracellular routing, i.e. additional nuclear localization sequences (NLS; the natural 23 kDa isoform and a 17D27R mutant) and/or a deletion of amino acids 26–29 (23A26–29 and 17Δ26–29), were introduced in basic fibroblast growth factor (bFGF). The mutants were assayed for their mitotic activity and their capacity to induce a tissue-specific response in human umbilical vein endothelial cells [HUVECs; induction of urokinase plasminogen activator receptor (u-PAR)], or in rat lens epithelial cells (fibre cell differentiation). In HUVECs, the 17D27R mutant had wild type activity, the 23 kDa and the Δ26–29 proteins were impaired in the induction of both mitosis and u-PAR. The Δ26–29 proteins, but not the 23 kDa protein or 17D27R mutant, were also impaired in receptor binding in that they bound only to a subset of receptors. The concentration of 17 kDa bFGF required for half maximal u-PAR response was 30 fold higher than for the half maximal ³H-thymidine incorporation. Addition of an NLS to bFGF strongly inhibited the induction of fibre cell differentiation, though it had little effect on the stimulation of DNA synthesis. The 17Δ26–29 kDa mutant had wild type differentiation activity but was a poor mitogen for lens epithelial cells.     

Differential Endothelial Migration and Proliferation to Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor

Abstract

Neovascularization is a feature of a variety of pathological processes. We compared the characteristics of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on migration and proliferation of human umbilical vein endothelium (HUVEC). Both VEGF and bFGF induced endothelial cell migration at similar concentrations (½ max. VEGF = ～1.0 ng/ml, bFGF = ～5.0 ng/ml). However, VEGF-stimulated migration was two-fold greater than bFGF at 1 and 10 ng/ml (p < 0.05). In contrast, bFGF induced proliferation four-fold more effectively than VEGF (½ max. 1 ng/ml and 1.4 ng/ ml respectively). Checkerboard migration assays for bFGF showed a predominantly chemokinetic pattern, whereas VEGF was predominantly chemotactic. VEGF and bFGF were not synergistic in monolayer proliferation and migration assays. Three angiogenesis inhibitors, alpha-interferon, TNP-470, and platelet factor-4, inhibited VEGF and bFGF induced cell migration. These results indicate that VEGF and bFGF are chemoattractants that stimulate endothelial migration by different mechanisms and that both can be inhibited by known angiogenesis inhibitors.     

Effects of Electromagnetic Field and Basic Fibroblast Growth Factor on Osteoblast＇s Growth

Osteoblasts of rat cultured in vitro were stimulated with pulsed 50 Hz electromagnetic field and basic fibroblast growth factor(bFGF). The MTT method, flow cytometry and histochemistry staining were used to detect cell proliferation, cell cycle and alkaline phosphatase. The results indicated : after stimulated by 1 mT electromagnetic field, the cells are more abundant,have more S phase percentages, 2 mT electromagnetic field have no evident effect on cells‘ growth;compared with electromagnetic field, the cells stimulated by bFGF are more abundant and have larger S phase ratios. Electromagnetic field and bFGF have no effect on cells, alkaline phosphatase. Therefore ,we concluded that electromagnetic field can enhance osteoblasts growth like some growth factor such as basic fibroblast growth factor, and the osteoblasts‘, characteristics was not changed.     

Bi-directional Signaling between Gastrointestinal Peptide Hormone Receptors and Epidermal Growth Factor Receptor

The mechanisms mediating the proliferative effects of gastrointestinal (GI) peptide hormones and their cognate G protein-coupled receptors are associated intimately with epidermal growth factor (EGF) receptor-regulated signaling pathways. Although transactivation of the EGF receptor is now recognized as a critical component in GI peptide hormone regulation of mitogenic signaling and cell migration, their interactions are far more complex and include potentiation of intracellular signaling pathways, regulation of ligand expression and release, and modulation of cell surface receptor expression. Mitogen-activated protein kinases play a central role integrating the signals from these receptor systems. This review summarizes the mechanisms that intertwine GI peptide hormone receptor- and EGF receptor-activation and functions.     

碱性成纤维细胞生长因子对成骨细胞生长与c-fos表达的影响

以重组牛碱性成纤维细胞生长因子 (rb- b FGF)刺激体外培养的大鼠成骨细胞 ,发现 :经过 1～ 2 d的刺激 ,成骨细胞产生很长的突起 ,细胞数量和活力较对照均有显著升高 ;经 rb- b FGF 1h的刺激后 ,c- fos基因的表达即明显高于对照 ,刺激 1～ 2 d后 c- fos基因的表达也明显高于对照。这表明 b FGF可促进大鼠成骨细胞增殖 ,提高c- fos基因的表达量。 c- fos表达量的提高 ,可能是各种刺激在促进细胞增殖的信号转化过程中的重要一步     

Differential Effects of an Antiserum to Epidermal Growth Factor on the Development of Transplanted Rat Embryos and Fetal Structures in Vivo

Abstract

In order to obtain information on the possible role of epidermal growth factor (EGF) in rat prenatal development, we tested the effects of a neutralizing antiserum to rat EGF and of recombinant human EGF on the growth and development of transplanted rat embryos and fetal structures. Ten-day embryos or 16-day fetal intestines (ileum and jejunum) or paws were transplanted under the capsule of both kidneys of young adult syngeneic host rats. Osmotic minipumps were used to infuse antiserum to rat EGF or normal rabbit serum (NRS) into the renal artery of the right kidney to ascertain direct effects on development. The transplants on the contralateral side served as internal controls. Infusion of the NRS did not affect growth of any of the fetal structures or of the embryo transplants. The antiserum to rEGF did not affect growth of the fetal ileum or paw transplants, but it inhibited growth of the fetal jejunum by 38%, and suppressed differentiation of hair follicles in the paws by ～90%. Tissue differentiation in the two segments of the intestine was unaffected by the antiserum. By contrast, growth of embryo transplants was stimulated by approximately 60% by the anti-EGF serum. Infusion of antiserum did not affect the growth of the kidneys upon which the transplants were grown, and infusion of different doses of recombinant human EGF had no effect on growth of embryo transplants.

Our data suggest that EGF may function as a negative growth regulator during the embryonic period, but it becomes a growth stimulator for specific tissues during the fetal period. These results are consistent with our previous findings with basic fibroblast and insulin-like growth factors in illustrating that the regulatory functions of growth factors may be more general during embryogenesis but they become more organ or tissue specific in the fetal period of development.     

成纤维细胞生长因子对多巴胺神经元的保护作用

目的:探讨成纤维细胞生长因子(FGF)对多巴胺能神经元的保护作用及其机制。方法:体外培养大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞),用6-羟基多巴胺(6-OHDA)和FGF分别或共处理PC12细胞,MTT法观察6-OHDA和FGF对PC12细胞活力的影响,流式细胞术观察PC12细胞凋亡率变化,TBA法测定PC12细胞丙二醛(MDA)含量变化。结果:6-OHDA处理后PC12细胞出现浓度依赖性活力下降,FGF能够减轻PC12细胞活力下降程度,降低细胞凋亡率,同时降低PC12细胞内MDA水平。结论:FGF对多巴胺能神经元具有保护作用,其机制与降低细胞内氧化应激水平有关。     

Differential Effects of Thalidomide on Angiogenesis and Tumor Growth in Mice

Thalidomide, clinically used as an antiinflammatory and antitumoral drug, inhibited sponge-induced angiogenesis when administered systemically (100 mg/kg^–1) in mice. However, it failed to inhibit solid Ehrlich tumor in the same mouse strain. We have used functional, biochemical and histological parameters to assess neovascularization and fibrovascular tissue infiltration of the mice sponge granuloma. The neovascularization growth as detected by development of blood flow and hemoglobin content extracted from the implants showed that thalidomide inhibited fibrovascular tissue formation by 40%. The functional and biochemical parameters correlated well with the histological study. Thalidomide had no inhibitory effect in the development of Ehrlich tumor. The detection of this selective action using the same animal strain bearing two different processes, supports the hypothesis that rather than species specificity, thalidomide is tissue specific. This approach may be used to identify the specificity of other therapeutic agents against distinct angiogenesis-dependent diseases.     

Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1^-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1^- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1⁺ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.     

Role of Epidermal Growth Factor Receptor Signaling in the Interaction of Neisseria meningitidis with Endothelial Cells

Neisseria meningitidis, the causative agent of meningitis and septicemia, attaches to and invades various cell types. Both steps induce and/or require tyrosine phosphorylation of host cell proteins. Here, we used a phospho array platform to identify active receptor tyrosine kinases (RTKs) and key signaling nodes in N. meningitidis-infected brain endothelial cells to decipher RTK-dependent signaling pathways necessary for bacterial uptake. We detected several activated RTKs, including the ErbB family receptors epidermal growth factor receptor (EGFR), ErbB2, and ErbB4. We found that pharmacological inhibition and genetic ablation of ErbB receptor tyrosine phosphorylation and expression resulted in decreased bacterial uptake and heterologous expression of EGFR, ErbB2, or ErbB4 in Chinese ovary hamster (CHO-K1) cells, which do not express of EGFR and ErbB4; the decrease caused a significant increase in meningococcal invasion. Activation of EGFR and ErbB4 was mediated by transactivation via the common ligand HB-EGF (heparin-binding EGF-like ligand), which was significantly elevated in infected cell culture supernatants. We furthermore determined that N. meningitidis induced phosphorylation of EGFR at Tyr845 independent of ligand binding, which required c-Src activation and was involved in mediating uptake of N. meningitidis into eukaryotic cells. Increased uptake was repressed by expression of EGFR Y845F, which harbored a point mutation in the kinase domain. In addition, activation of ErbB4 at its autophosphorylation site, Tyr1284, and phosphorylation of ErbB2 Thr686 were observed. Altogether, our results provide evidence that EGFR, ErbB2, and ErbB4 are activated in response to N. meningitidis infection and shed new light on the role of ErbB signaling in meningococcal infection biology.     

Protective Effects of Basic Fibroblast Growth Factor in Early Atherosclerosis

Objectives: The role of basic fibroblast growth factor (bFGF) in atherosclerotic plaque formation is incompletely understood. Although it may act as a proatherogenic factor due to its stimulatory effect on smooth muscle cell growth, previous studies have suggested that it may also have beneficial effects by reversing endothelial dysfunction in experimental models. Our purpose was to evaluate the effects of systemic chronic administration of basic FGF on the development of atherosclerotic plaques in a rabbit model. Method: We investigated the effect of bFGF or placebo (2.5 μg IV twice a week), begun on the same day as a cholesterol (2%) diet, and continued for 5 or 10 weeks, on in vitro reactivity, vascular cell adhesion molecule-1 (VCAM-1) expression and plaque development (protocol 1; [Formula: See Text]). The effects of bFGF or placebo (2.5 μg IV once a week) were also studied in animals fed a 0.2% cholesterol diet and sacrificed at 3 months (protocol 2; [Formula: See Text]). Results were compared to those of rabbits fed with a normal chow (normal animals). Results: In protocol 1, bFGF administration for 5 weeks was associated with an improvement in endothelial function [Formula: See Text] with a decrease in VCAM-1 expression [Formula: See Text] and in the macrophage content of the plaque [Formula: See Text] This preventive effect was lost at 10 weeks. In protocol 2, bFGF was associated with similar "beneficial" endpoints as observed at 5 weeks in protocol 1. Conclusions: Administration of bFGF is associated with important beneficial structural and functional effects in the early stage of experimental atherosclerosis. These results may help us to understand the role of growth factors in atherosclerosis and to anticipate their effects in human arteries.     

Prediction of Growth Factor Effects on Engineered Cartilage Composition Using Deterministic and Stochastic Modeling

In the design of engineered tissues, guided balance of biomaterial degeneration with tissue synthesis offers refined control of construct development. The objective of this study was to develop a mathematical model that describes the steady state metabolism of extracellular matrix molecules (ECM: glycosaminoglycan and collagen) in an engineered cartilage construct taking into account localized environmental changes that may arise because of the application of growth factors. The variable effects of growth factors were incorporated in the form of random noise rather than the difference in rates of synthesis and catabolism. Thus, the frequency of ECM accumulation for each matrix molecule in the steady state under the random influence of growth factor was produced relative to the matrix carrying capacity. Published synthesis-rate time constants and steady state ECM conditions from chondrocyte-polymer scaffold composites provided both input and validation for the model. Although the presence of growth factors in the presented system dynamics were considered randomized, the results described a positive feedback or promotional ECM synthesis at low levels of growth factors. While a negative feedback or inhibition of ECM synthesis was characterized at higher levels of growth factors. This transition phenomenon is based on a comparison with the results of a steady state condition in the form of a deterministic model and supports previous reports of guided accumulation in musculoskeletal, connective, and neuronal tissues.