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.     

Mitogenic growth factors regulate differentially early gene mRNA expression: a study on two clones of 3T3 fibroblasts.

The relationship between cell proliferation and mRNA levels of the immediate early genes c-fos, c-jun, and jun B has been investigated in two clones of 3T3 fibroblasts (D1-3T3 and N2-3T3) upon treatment with basic fibroblast growth factor (bFGF), thrombin, phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (Bt2cAMP). The 3T3-derived clone D1-3T3 almost stops dividing upon serum deprivation, while the N2-3T3 clone does not. The proliferation of the two clones was stimulated by thrombin and PMA and inhibited by Bt2cAMP. Basic FGF stimulated the growth of D1-3T3 but partly inhibited that of N2-3T3 cells. In spite of variable mitogenic response, immediate early genes, c-fos, c-jun, jun B, and c-myc, were induced by the growth factors and by PMA in both cell clones. In our experimental conditions the early gene mRNAs were expressed independently; i.e., the expression of one protooncogene had no bearing on the expression of the other. The cell growth was not directly related to the expression of a particular protooncogene mRNA. Data are presented showing that early gene mRNA expression induced by bFGF or thrombin was not mediated by protein kinase C activation while thrombin-induced mitosis was. Basic FGF induced a part of c-jun mRNA expression, but not mitosis, through a pertussis toxin-sensitive mechanism.     

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.     

Inhibition of insulin-like growth factor-I mitogenic action by zinc chelation is associated with a decreased mitogen-activated protein kinase activation in RAT-1 fibroblasts.

The mechanisms responsible for the resistance to the anabolic actions of IGF-I induced by zinc deficiency are not understood. We showed that zinc chelation by DTPA (diethylenetriaminepenta-acetic acid) inhibits [3H]thymidine incorporation stimulated by IGF-I in Rat-1 fibroblasts. This inhibition was specific of zinc chelation since it was prevented by the addition of zinc to DTPA. The stimulation of MAPK, which is crucial for the [3H]thymidine incorporation induced by IGF-I in Rat-1 cells, was partially blunted by DTPA. Therefore, the inhibition of the mitogenic action of IGF-I in Rat-1 fibroblasts by DTPA is potentially caused by decreased MAPK activation by IGF-I.     

Demonstration of L1-related mRNAs in rat brain using DNA oligonucleotide probes.

Only one copy of the cell adhesion molecule L1 gene is present in the mouse genome, and only one mRNA of 6 kilobases (kb) is expressed in mouse brain [1987, Neurosci. Lett. 82, 89-94]. We have constructed 5 synthetic oligonucleotide probes covering different parts of the published mouse L1 cDNA sequence. Using these probes 3 distinct mRNAs of 9.0, 7.0 and 6.0 kb in rat brain could be demonstrated. Hybridizations performed at different stringency conditions indicated that the 9.0 and 7.0 kb mRNAs were highly related to the L1 mRNA of 6.0 kb expressed in rat brain. The 7.0 kb mRNA is possibly coding for a rat homologue of chicken Nr-CAM, whereas the 9.0 kb mRNA may code for a new member of the L1 family.     

Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.     

Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels.

Fibroblasts represent one of the in vivo sites of insulin-like growth factor-I (IGF-I) production. In this study rat dermal fibroblasts in culture were used as a model system to assess the effect of activation of protein kinase-C on the levels of the mRNAs encoding IGF-I and another growth factor, basic fibroblast growth factor (bFGF). IGF-I and bFGF mRNA levels were determined using a solution hybridization/RNase protection assay. Treatment of cells in serum-free medium containing 0.25% BSA (MEM + BSA) with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) decreased IGF-I and increased bFGF mRNA levels in a time- and dose-dependent fashion. The peak effect of 100 nM PMA on IGF-I mRNA levels occurred at 9 h, whereas the peak effect on bFGF mRNA levels occurred after 3 h of incubation. In dose-response studies, half-maximal inhibition of IGF-I mRNA levels was achieved with approximately 0.08 nM PMA, while half-maximal stimulation of bFGF mRNA levels was achieved with approximately 3 nM PMA. Inhibition of protein synthesis with cycloheximide abrogated the effect of PMA on bFGF mRNA levels, but only partially inhibited the effect of PMA on IGF-I mRNA levels. Studies employing sphingosine or staurosporine to inhibit protein kinase-C or preincubation in high doses of PMA to down-regulate protein kinase-C suggested that the effect of PMA on IGF-I and bFGF mRNA levels was mediated by activation of protein kinase-C, although both staurosporine and sphingosine had independent effects on the levels of these mRNAs and down-regulation of protein kinase-C had a sustained effect on IGF-I mRNA levels. Ligands known to activate protein kinase-C were then tested. Treatment of cells with 100 micrograms/ml of the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol decreased IGF-I mRNA levels to 25% and increased bFGF mRNA levels to 520% of the level present in cells maintained in MEM + BSA. Treatment of cells with thrombin or bradykinin also decreased IGF-I mRNA levels and increased bFGF mRNA levels, but whereas the effect of thrombin on IGF-I mRNA levels was marked, the effect of bradykinin was minimal, and whereas the effect of thrombin on bFGF mRNA levels was sustained, the effect of bradykinin was transient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C.

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.