Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.     

The mitogenic signaling pathway but not the plasminogen activator- inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells

Basic fibroblast growth factor (bFGF) induces cell proliferation and plasminogen activator (PA) activity in transformed fetal bovine aortic endothelial (FBAE) GM 7373 cells. A similar response is observed after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In these cells, bFGF and TPA cause activation of protein kinase C (PKC), as demonstrated by the induction of the phosphorylation of an 87-kD PKC substrate in intact cells and by the increase in membrane-associated PKC activity. Activation of PKC by bFGF or TPA is inhibited in cells made PKC-deficient by pretreatment with high concentrations of TPA. The mitogenic activity of bFGF or of TPA is completely inhibited in PKC- deficient cells or in naive cells treated with the PKC inhibitor H-7. However, these cells proliferate in response to serum, epidermal growth factor, and dibutyryl cyclic AMP. Similar results are obtained in normal FBAE AG 7680 cells. These data indicate that activation of PKC is responsible for the mitogenic activity of bFGF in FBAE cells. On the contrary, the PA-inducing activity of bFGF is unaffected by down- regulation of PKC or by treatment with the PKC inhibitor H-7 in both transformed GM 7373 and normal AG 7680 cells. bFGF induces a rapid 45Ca influx in naive and in PKC-deprived GM 7373 cells. In these cells, addition of EGTA to the incubation medium prevents both the 45Ca influx and the increase in PA activity induced by bFGF, without affecting its mitogenic activity. Even though the involvement of PKC in the increase of cell-associated PA activity induced by bFGF can not be completely dismissed, the present results suggest a role of calcium entry in the modulation of the PA-inducing activity of bFGF.     

Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells

A mitogenic and plasminogen activator (PA)-inducing activity for endothelial cells has been identified in serum-free culture medium of normal AG 7680 and transformed tumorigenic GM 7373 fetal bovine aortic endothelial (FBAE) cells. The activity binds to heparin-Sepharose and it is quenched by polyclonal anti-human placental basic fibroblast growth factor (bFGF) antibodies. In the serum-free conditioned medium of FBAE cells, the anti-bFGF antiserum recognizes an immunorective Mr 20,000 molecule which co-purifies with the mitogenic and PA-inducing activity on a heparin-Sepharose column. The partially purified Mr 20,000 bFGF-like molecule competes with the typical Mr 18,000 125I-bFGF form for the binding to high-affinity bFGF receptors in intact GM 7373 cells. Immunoprecipitation of biosynthetically labeled GM 7373 cells with anti-bFGF antiserum confirms the presence of a Mr 20,000 bFGF-like molecule in the conditioned medium of these cells and identifies the typical Mr 16,000 and Mr 18,000 bFGF forms and two high-molecular-weight immunoreactive Mr 22,000 and Mr 25,000 bFGF forms in their cell extract. Immunoreactive Mr 20,000 bFGF is detectable also in the conditioned medium of transformed nontumorigenic FBAE GM 7372 cells and of adult bovine aortic endothelial cells, but not in the culture medium of nonendothelial cell types, including rat and mouse fibroblasts, human hepatoma, and human endometrial adenocarcinoma cells. The results indicate that bovine endothelial cells secrete a Mr 20,000 bFGF-like molecule which shares several biological, biochemical, and immunological characteristics with the typical cell-associated Mr 18,000 bFGF.     

Basic fibroblast growth factor: production, mitogenic response, and post-receptor signal transduction in cultured normal and transformed fetal bovine aortic endothelial cells

Normal FBAE AG 7680 cells and chemically transformed FBAE GM 7373 cells were compared for their capacity to produce and to respond to bFGF. Normal FBAE cells showed higher levels of bFGF protein and of poly(A)+ bFGF mRNA than transformed GM 7373 cells, indicating that chemical transformation in FBAE cells is paralleled by a decrease of bFGF gene expression. Basic FGF induced cell proliferation in both normal and transformed FBAE cells. However, bFGF appeared to be much more potent in transformed than in normal cells. No differences in bFGF membrane receptors were observed between normal and transformed FBAE cells in terms of apparent molecular weight, number per cell, dissociation constant, and kinetic of downregulation. In respect to normal cells, however, transformed GM 7373 cells showed higher basal levels of PKC activity. This kinase is activated by bFGF and is involved in mediating the mitogenic activity of bFGF, as shown by the capacity of the PKC inhibitor H-7 to abolish the mitogenic activity of bFGF both in normal and transformed FBAE cells. Like bFGF, the PKC activators DAG and TPA exerted a stronger mitogenic activity in transformed than in normal FBAE cells. Thus, the different susceptibility of normal and transformed FBAE cells to bFGF appears to depend on differences in the post-receptor signal transduction mediated by PKC rather than on differences in bFGF receptors. The results indicate that chemical transformation causes significant modifications of bFGF physiology in FBAE cells. The relevance of these modifications to the genesis of tumors of vascular origin deserves further investigation.     

Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells.

Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two molecules require 16 and 6 h, respectively, of activation of PKC to induce 50% of maximal cell proliferation. The requirement for a long-lasting activation of PKC appears to be a mechanism for the control of cell proliferation capable of discriminating among transient nonmitogenic stimuli and long-lasting mitogenic stimuli.     

Purification of basic fibroblast growth factor from rat brain: identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.     

Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form

Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECM-bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF.     

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.     

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.     

Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: Biological activity and intracellular fate of human recombinant Mr 24,000 bFGF

The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M_r) of 24kD, 22.5 kD, 22kD, and 18kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF, cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 13-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products. In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells. © 1994 Wiley-Liss, Inc.     

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.     

Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver

A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.     

Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells.

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta-type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (³H)-arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo-(2-³H)-inositol nor did it induce calcium mobilization, as measured by fura-2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF-induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.     

1,2-Dioctanoyl-glycerol induces a discrete but transient translocation of protein kinase C as well as the inhibition of MCF-7 cell proliferation

Exposure of MCF-7 human breast cancer cells to phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) results in a dose-dependent inhibition of cell proliferation. One of the earliest biochemical events induced by TPA is the translocation of protein kinase C from the cytosolic to the particulate compartment. We have investigated the effects of permeant diacylglycerol 1,2-dioctanoyl-glycerol (diC8) on both protein kinase C activity and MCF-7 cell proliferation. DiC8 induces a discrete but significant translocation of protein kinase C within the first minutes of MCF-7 cell treatment (26 +/- 6%, mean +/- SD of 5 different experiments, upon 5 min incubation in the presence of 43 micrograms/ml diC8). However, this effect is only transient as the enzymatic activity returns to the control value after 60 min. DiC8 mimics the effect of TPA on MCF-7 cell proliferation. The dose-response curves for both protein kinase C translocation and cell growth inhibition show that diC8 exerts its effects on both parameters in the same range of concentrations, despite some discrepancies at the lowest doses. We also report that long-term treatment of the cells with diC8 does not lead to the protein kinase C disappearance observed during prolonged exposure to TPA. All together, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation and suggest that the enzyme translocation but not its down-regulation could be a pre-requisite in the biological cell response.     

Modulation of endothelial cell proliferation,adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin

Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence -Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependet and reversible. Thus, the antiproliferative activities of the thrombospondin peptides and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.     

Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation.     

Protamine sulfate inhibits mitogenic activities of the extracellular matrix and fibroblast growth factor, but potentiates that of epidermal growth factor

Protamine sulfate, an inhibitor of angiogenesis in vivo, markedly inhibits the ability of angiogenic factors such as acidic or basic fibroblast growth factor (aFGF, bFGF) to stimulate the proliferation in vitro of either BHK-21 cells or vascular endothelial cells. The inhibition is reversible, and cells remain viable even after prolonged exposure to protamine sulfate. Protamine sulfate inhibits the mitogenic effects of both growth factors by preventing them from binding to their common cell surface receptors. It also inhibits the mitogenic activity of the extracellular matrix produced by bovine corneal endothelial cells. This substrate has been shown in previous studies to replace the requirement for FGF of many cell types. In contrast, protamine sulfate potentiates the mitogenic activity of epidermal growth factor (EGF). This indicates that protamine sulfate also acts at cellular sites which are not associated with FGF receptors.