Dura mater biology: autocrine and paracrine effects of fibroblast growth factor 2

The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells.Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.     

Mechanical stress induces DNA synthesis in PDL fibroblasts by a mechanism unrelated to autocrine growth factor action

Periodontal regeneration is thought to require the proliferation of stress-sensitive periodontal ligament (PDL) fibroblast cells. The influence of physiological amounts of mechanical stretching on the DNA synthesis potential of human PDL fibroblasts was examined by means of an established, simple in vitro system of stretch application. A significant increase in the relative levels of incorporation of tritiated thymidine was observed in cultures stretched for 1–6 h. Neutralising antibodies for platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β) did not blunt the DNA synthesis induction. This mitogenic response to stretch appears to be independent of an autocrine mechanism involving growth factors in general, because stretch-conditioned medium, when transferred to non-stretched fibroblasts, did not mimic the mitogenic effect of stretch.     

Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115.

An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)-like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC-3 cells in an autocrine mechanism. Since the androgen-induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC-3 cells. Heparin was found to exhibit significant inhibition of testosterone-induced growth in a concentration-dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 micrograms/ml. DNA synthesis of SC-3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC-3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen-induced growth factor in the conditioned medium from testosterone-treated cells. These results indicate that heparin inhibits autocrine loop in SC-3 cells induced by androgen. Thus, the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan.     

Alpha-tocopheryl succinate induces cytostasis and apoptosis in osteosarcoma cells: the role of E2F1

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analog of vitamin E, induces cell cycle arrest, differentiation, and triggers apoptosis. We examined the ability of alpha-TOS to induce cytostasis and/or apoptosis in two human osteosarcoma cell lines, which carry wild-type pRb but differ in the p53 status. In the wt-p53 cells, alpha-TOS induced apoptosis, which was associated with p53 activation and enhanced E2F1 expression. Mutant p53 cells failed to undergo apoptosis when challenged with alpha-TOS. The cell growth arrest after alpha-TOS treatment was associated with a reduced expression of E2F1. Knocking down E2F1 rendered the alpha-TOS-sensitive cells rather resistant to the apoptotic stimulus inducing a marked and prolonged cell growth arrest. We conclude that alpha-TOS induces cell growth arrest or apoptosis involving E2F1.     

A truncated form of fibroblast growth factor receptor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor.

A truncated form of the type 1 fibroblast growth factor receptor (FGFR1) lacking most of its cytoplasmic domain was tested for its ability to inhibit signal transduction by each of three different wild-type FGFRs (FGFR1, 2, and 3). When the truncated FGFR1 was expressed in Xenopus oocytes in excess of each wild-type FGFR, mobilization of intracellular calcium mediated by the wild-type FGFRs was completely blocked. The truncated FGFR did not inhibit signal transduction by the co-expressed platelet-derived growth factor beta-receptor. A form of truncated FGFR1 which lacked the first immunoglobulin-like domain also inhibited signal transduction by wild-type FGFRs. Truncated FGFR formed complexes with wild-type FGFR in the presence of basic FGF in intact cells. These observations were consistent with the hypothesis that the truncated FGFR interacted with wild-type FGFRs to form nonfunctional heterodimers, thus eliminating the signaling by the wild-type FGFRs. The observation that signaling by multiple types of FGFR can be blocked by a single type of truncated FGFR suggests that the different types of FGFR can interact with each other in ligand-mediated complexes. These findings provide a molecular basis for inhibiting the actions of FGFs in vivo.     

Androgen-induced growth factor and its receptor: Demonstration of the androgen-induced autocrine loop in mouse mammary carcinoma cells

SC-3 cells derived from mouse mammary carcinoma (Shinogi carcinoma 115) exhibit remarkable growth enhancement and cell morphology change in response to androgen stimuli. These events are mediated through an androgen-induced growth factor (AIGF). Amino acid sequence deduced from cDNA reveals that AIGF has 215 amino acids with a signal peptide and scattered regions homologous to fibroblast growth factor (FGF) family proteins. The biological ability of AIGF to stimulate SC-3 cell growth is inhibited by heparin or suramin. More importantly, antisense oligodeoxynucleotide of AIGF can block androgen-induced growth of SC-3 cells. Upon synthesis under the control of androgen, AIGF is immediately secreted into the extracellular space without intracellular accumulation. At the early phase (18–24 h) of androgen stimulation, however, AIGF is mainly associated with the glycosaminoglycan on the cell surface or extracellular matris. In addition, treatment of SC-3 cells with sulfation blocker (chlorate) or heparitinase results in the abolishment of their ability to respond to androgen or AIGF, indicating that heparan sulfate has important roles for condensing AIGF on or near cell surface as well as potentiating the biological activity of AIGF. Then, AIGF can bind to the FGF receptor. Northern blot analysis and cDNA cloning indicate that SC-3 cells predominantly express the FGF receptor 1 with some altered amino acid sequences. Transfection of expression vectors of AIGF and this variant from of FGF receptor 1 into FGF receptor-negative myoblast cells (L 6 cells) confirms that a variant from of FGF receptor 1 is a receptor of AIGF. These results clearly demonstrate that an autocrine mechanism is operating in androgen-induced growth of SC-3 cells.     

Suppression of basic fibroblast growth factor expression by antisense oligodeoxynucleotides inhibits the growth of transformed human astrocytes.

Basic fibroblast growth factor (bFGF) is a heparin-binding protein expressing potent mitogenic and angiogenic properties. Elevated levels of bFGF have recently been described in human glioma cell lines. The high degree of vascularity and invasiveness which characterize human gliomas suggest that activated expression of bFGF or similar proteins may be related to the aberrant growth patterns of these tumors. The influence of endogenous bFGF on glioma cell growth in vitro was evaluated in the present study by down-regulating bFGF expression using antisense oligonucleotide primers. The addition of 50 microM bFGF-specific antisense primer to the human glioma cell line SNB-19 resulted in an 80% inhibition in glioma growth. This effect was saturable and specific. Antisense primers directed to two different sites of bFGF mRNA were effective in suppressing SNB-19 growth, whereas sense strand primer was ineffective. Furthermore, only the antisense primer significantly reduced the specific activity of bFGF protein in SNB-19 cell extracts. Neither antisense or sense primers inhibited the growth of non-transformed human glia. bFGF mRNA was detected in both transformed and non-transformed human glia by polymerase chain reaction analysis suggesting that alterations in bFGF isoform content or activity may be specifically related to abnormal growth control in human gliomas.     

Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.     

Bronchial epithelial cell growth regulation in fibroblast cocultures: the role of hepatocyte growth factor

Proliferation of bronchial epithelial cells is an important biological process in physiological conditions and various lung diseases. The objective of this study was to determine how bronchial fibroblasts influence bronchial epithelial cell proliferation. The proliferative activity in cocultures was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct cells counts. Concentration of cytokines was measured in cell culture supernatants by means of ELISA. In primary cell cocultures, fibroblasts or fibroblast-conditioned medium enhanced 1.85-fold the proliferation of primary bronchial epithelial cells (P < 0.02) compared with bronchial epithelial cells cultured alone. The proliferative activity in cocultures and in fibroblast-conditioned medium was reduced by neutralizing antibody to hepatocyte growth factor (HGF) and HGF receptor c-met. Neutralizing antibodies to FGF-7 and IGF-1 had no effect. Treatment of fibroblast-epithelial cocultures with anti-IL-6 and anti-TNF-alpha neutralizing antibodies and with indomethacin decreased production of HGF. These results indicate that cytokines and PGE(2) may indirectly mediate epithelial cell proliferation via the regulation of HGF in bronchial stromal cells and that HGF plays a crucial role in proinflammatory cytokine-induced proliferation in the experimental system studied.     

Modulation of the epidermal growth factor receptor by basic fibroblast growth factor

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibited of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled. © 1993 Wiley-Liss, Inc.     

Sodium ascorbate and basic fibroblast growth factor protect muscle-derived cells from H2O2-induced oxidative stress

Engraftment of muscle-derived cells (MDCs) into the urethral sphincter may cure urinary incontinence. However, poor cell survival after injection prompted us to evaluate whether 24-h preincubation with sodium ascorbate (ASC) or basic fibroblast growth factor (bFGF) prior to cell transfer improves the survival of MDCs facing oxidative stress in vitro. We examined MDCs isolated from female rats for the presence of myogenic markers and for the ability to differentiate and respond to growth factors. Isolated cells were positive for desmin, M-cadherin, and myogenin. The fusion index exceeded 29%, and Akt kinase was phosphorylated at Ser473 residue upon exposure to insulin-like growth factor 1 (100 ng/ml). We then autologously transplanted MDCs transfected with lacZ marker gene into urethral wall of the rats; 2 wk later, the urethra and caudal wall of the urinary bladder were harvested from these animals. Serial cryosections revealed that transplanted cells formed multinuclear clusters at injection sites. In addition, we found that the viability of MDCs exposed to a cytotoxic concentration of H2O2 was higher after preincubation with 0.1 mM ASC (2.6-fold), 10 ng/ml bFGF (2.9-fold), or 20 ng/ml bFGF (3.5-fold) than that after exposure to H2O2 only. We conclude that preincubation with ASC or bFGF increases the resistance of MDCs to oxidative stress in vitro. Pretreatment with either agent might be used to enhance survival of MDCs after transplantation.     

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997. © 1997 Wiley-Liss, Inc.     

The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies

Basic fibroblast growth factor/fibroblast growth factor-2 is one of the best characterized of the pro-angiogenic cytokines. This review describes its history, as well as its role in tumor angiogenesis associated with haematological malignancies, as traced by the main contributions to the international medical literature.     

Sef inhibits fibroblast growth factor signaling by inhibiting FGFR1 tyrosine phosphorylation and subsequent ERK activation

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration as well as differentiation events such as angiogenesis, osteogenesis, and chondrogenesis. Recently, genetic screens in Drosophila and gene expression screens in zebrafish have resulted in the identification of several feedback inhibitors of FGF signaling. One of these, Sef (similar expression to fgf genes), encodes a transmembrane protein that belongs to the FGF synexpression group. Here we show that like zebrafish Sef (zSef), mouse Sef (mSef) interacts with FGFR1 and that the cytoplasmic domain of mSef mediates this interaction. Overexpression of mSef in NIH3T3 cells results in a decrease in FGF-induced cell proliferation associated with a decrease in Tyr phosphorylation of FGFR1 and FRS2. As a consequence, there is a reduction in the phosphorylation of Raf-1 at Ser(338), MEK1/2 at Ser(217) and Ser(221), and ERK1/2 at Thr(202) and Tyr(204). Furthermore, mSef inhibits ERK activation mediated by a constitutively activated FGFR1 but not by a constitutively active Ras and decreases FGF but not PDGF-mediated activation of Akt. These results indicate that Sef exerts its inhibitory effects at the level of FGFR and upstream of Ras providing an additional level of negative regulation of FGF signaling.     

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.     

Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. Differences to the activation by IL-1

The pronounced synovial hyperplasia often found in the joints of patients with rheumatoid arthritis could be explained partially by the action of monocyte-macrophage polypeptides (monokines). This report demonstrates that two cytokines which may be derived from monocyte-macrophage populations, namely platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), stimulate the DNA synthesis and proliferation of human synovial fibroblast-like cells cultured in low (i.e., 1%) fetal bovine serum. Epidermal growth factor, insulin-like growth factor-I, insulin-like growth factor-II (multiplication stimulating activity) and substance P were inactive. Unlike IL-1, PDGF and FGF do not also stimulate PGE2, plasminogen activator, and hyaluronic acid levels. Thus PDGF and FGF, arising from stimulated monocyte-macrophages, may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory arthritic disease. The synovial cells respond to a variety of cytokines in different ways suggesting multiple-signaling pathways.