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1.
The fibroblast growth factor (FGF) receptor of human umbilical vein-derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal growth factor, insulin, or transferrin. The lectin concanavalin-A does not inhibit the binding of 125l-bFGF to HUE cell-surface receptors, whereas it inhibits bFGF binding to BHK-21 cell-surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti-angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor. 相似文献
2.
Brain-derived growth factor is a chemoattractant for fibroblasts and astroglial cells 总被引:5,自引:0,他引:5
R M Senior S S Huang G L Griffin J S Huang 《Biochemical and biophysical research communications》1986,141(1):67-72
Bovine brain-derived growth factor (BDGF), a 16-17 kDa protein with biochemical properties resembling brain-derived acidic fibroblast growth factor (acidic FGF) and endothelial cell growth factor, was found to have potent chemotactic activity for bovine ligament fibroblasts, human skin fibroblasts and rat astroglial cells, maximal at 100-200 pg/ml. The chemotactic activity was completely blocked by protamine sulfate (5 ug/ml), an inhibitor of receptor-binding and mitogenic activity of BDGF. BDGF did not stimulate migration of human monocytes. These results indicate that the effects of BDGF 'in vivo' might extend to mesenchymal cell recruitment. 相似文献
3.
Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity 总被引:2,自引:0,他引:2
Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells. 相似文献
4.
Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted 总被引:20,自引:0,他引:20
I Vlodavsky R Fridman R Sullivan J Sasse M Klagsbrun 《Journal of cellular physiology》1987,131(3):402-408
Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall. 相似文献
5.
Jun-Hyeog Jang Fen Wang Mikio Kan 《In vitro cellular & developmental biology. Animal》1997,33(10):819-824
Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute
a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates
and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate
the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein,
protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to
FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing
the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect
cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan
sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations
in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent
heparan sulfate chains required for assembly and activation of the FGF signal transduction complex. 相似文献
6.
J Rosenbaum P Mavier A M Preaux D Dhumeaux 《Biochemical and biophysical research communications》1989,164(3):1099-1104
We have investigated whether isolated mouse hepatic sinusoidal endothelial cells (HSEC) synthesized basic FGF. HSEC lysate was fractionated by heparin-Sepharose chromatography. A peak of mitogenic activity for Balb/c 3T3 fibroblasts was eluted with 3M NaCl. Several arguments suggested that the mitogenic factor was related to bFGF: a) its affinity for heparin; b) the loss of its mitogenic activity by heating at 65 degrees C, which was prevented in the presence of heparin; c) the abolition of its mitogenic activity in the presence of protamine sulfate; d) finally, its mitogenic effect was reduced in the presence of antibody to bFGF. These data demonstrate the presence of a bFGF-like molecule in HSEC. This molecule could be involved in the regulation of the neighboring Ito cell proliferation. 相似文献
7.
Role of protein kinase C in the inhibition by fibroblast growth factor of apoptosis in serum-depleted endothelial cells 总被引:7,自引:0,他引:7
S Araki Y Simada K Kaji H Hayashi 《Biochemical and biophysical research communications》1990,172(3):1081-1085
Apoptosis in vascular endothelial cells is suppressed by fibroblast growth factor (FGF)1. In order to investigate the signal transduction system that regulates endothelial apoptosis, we studied the effects of several mitogenic factors. Apoptosis occurred in human vascular endothelial cells under serum-free conditions, and FGF inhibited apoptosis without a requirement of any cooperative factors, as distinct from the mitogenic response. Other mitogenic agents, such as epidermal growth factor, transferrin, transforming growth factor beta, and interleukin 1 etc., with the exception of dexamethasone, had no such inhibitory effects. The effect of FGF was mimicked by a phorbol ester and was prevented by an inhibitor of protein kinase C. The results suggest that the FGF and protein kinase C are important in endothelial apoptosis. 相似文献
8.
《Matrix biology》2014
Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α2β1 integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels. 相似文献
9.
W Greil M Rafferzeder G Bechtner R G?rtner 《Molecular endocrinology (Baltimore, Md.)》1989,3(5):858-867
It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors. 相似文献
10.
Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules 总被引:44,自引:0,他引:44
Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response. 相似文献
11.
Anthon du P. Heyns Amiram Eldor Israel Vlodavsky Nurit Kaiser Rafael Fridman Amos Panet 《Experimental cell research》1985,161(2):297-306
We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFN alpha, recombinant human IFN alpha 2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent. 相似文献
12.
A growth factor has been purified to homogeneity from human pituitary glands. The pituitary growth factor (PGF) is trypsin-sensitive and acid- and heat-labile and has a molecular weight of 18,000 and an isoelectric point of 7.5. PGF was purified by heparin and copper affinity chromatography followed by carboxymethylcellulose 52. The amino-terminal amino acid sequence of PGF was established as PALPEXGGXGA and is identical with that of basic fibroblast growth factor at the identified amino acid residues. PGF was mitogenic for rabbit fetal chondrocytes and bovine corneal endothelial cells in the range of 0.015-15 ng mL-1. Heparin alone at low concentrations (0.5 microgram mL-1) was found to be weakly mitogenic for rabbit fetal chondrocytes. In combination with PGF a marked increase in cell growth was observed, which was inhibited by protamine sulfate. These data demonstrate the presence of a potent mitogen in human pituitaries that is structurally related to basic fibroblast growth factor and synergizes with heparin to promote cell growth. 相似文献
13.
Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors 总被引:26,自引:0,他引:26
D P Bottaro J S Rubin D Ron P W Finch C Florio S A Aaronson 《The Journal of biological chemistry》1990,265(22):12767-12770
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF. 相似文献
14.
In vitro regulation of pig Sertoli cell growth and function: effects of fibroblast growth factor and somatomedin-C 总被引:2,自引:0,他引:2
The effects of insulin, somatomedin-C (Sm-C), epidermal growth factor (EGF), fibroblast growth factor (FGF), vitamin E, and retinoic acid on growth and function of immature cultured pig Sertoli cells were investigated. All these factors, except vitamin E, stimulated Sertoli cell DNA synthesis and proliferation. The mitogenic effects of insulin observed only at micromolar concentrations were similar to those induced by nanomolar concentrations of Sm-C or EGF, but significantly less than those induced by FGF. The effects of EGF and Sm-C were almost additive, whereas those of Sm-C and FGF were synergistic. After a 6-day treatment, FGF and retinoic acid induced a significant increase in the number of follicle-stimulating hormone (FSH) receptors per cell, and in FSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Sm-C, which alone had no effect on these two parameters, potentiated FGF action. Basal plasminogen activator activity was enhanced after the 6-day treatment with EGF plus insulin and, particularly, with FGF plus insulin. Similarly, the response of the latter group to FSH was significantly higher than in any other group of cells. FGF was also able to stimulate cell multiplication and enhanced the FSH receptor number of Sertoli cells isolated from 15- and 26-day-old rats. Thus, FGF is the most potent known mitogenic factor for cultured Sertoli cells, and it stimulates the phenotypic expression of these cells. 相似文献
15.
Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis 总被引:63,自引:25,他引:38
We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells. 相似文献
16.
Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)-beta, fibroblast growth factor (FGF), and insulin-like growth factor I (IGF-I). In serum-free defined medium the following observations were made: TGF-beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF-I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF-beta could not be counteracted by any combination of IGF-I or FGF. The proliferation-depressing activity of TGF-beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF-I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF-I, alone. By altering the concentrations of TGF-beta, FGF, and IGF-I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent. 相似文献
17.
Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma. 相似文献
18.
Qiao D Meyer K Mundhenke C Drew SA Friedl A 《The Journal of biological chemistry》2003,278(18):16045-16053
Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy. 相似文献
19.
Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells 总被引:13,自引:4,他引:9
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The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF. 相似文献
20.
Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mammalian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl-phosphatidylinositol (Pl) residue, which can be released by treatment with a glycosyl-Pl specific phospholipase C (Pl-PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to Pl-PLC resulted in release of cell surface-associated, growth-promoting activity that was neutralized by antibasic fibroblast growth factor (bFGF) antibodies. Active bFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK-21, NIH/3T3, PF-HR9) that expressed little or no bFGF, as opposed to Pl-PLC-mediated release of active bFGF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant bFGF in a radioreceptor assay. Addition of Pl-PLC to sparsely seeded vascular endothelial cells resulted in a marked stimulation of cell proliferation, but there was no mitogenic effect of Pl-PLC on 3T3 fibroblasts. Studies with exogenously added 125I-bFGF revealed that about 6.5% and 20% of the cell surface-bound bFGF were released by treatment with Pl-PLC and heparitinase, respectively. Both enzymes also released sulfate-labeled heparan sulfate from metabolically labeled 3T3 fibroblasts. Pl-PLC failed to release 125I-bFGF from the subendothelial extracellular matrix (ECM), as compared to release of 60% of the ECM-bound bFGF by heparitinase. Our results indicate that 3-8% of the total cellular content of bFGF is associated with glycosyl-Pl anchored cell surface HSPG. This FGF may exert both autocrine and paracrine effects, provided that it is released by Pl-PLC and adequately presented to high affinity bFGF cell surface receptor sites. 相似文献