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D M Ornitz

Publications and source records attributed to D M Ornitz.

At least 55 records · Page 3Linked to original sources
PubMed1997

Heparin-induced self-association of fibroblast growth factor-2. Evidence for two oligomerization processes.

Fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, requires heparin for dimerization and activation of the FGF receptor tyrosine kinase. The binding of multiple fibroblast growth factors by heparin may be necessary for dimerization of the FGF receptor. Analytical ultracentrifugation of FGF-2 in the presence of heparin-derived saccharides shows that both an active heparin octasaccharide and an inactive heparin-like disaccharide induce fibroblast growth factor-2 self-association. Analysis of the data indicates that the heparin octasaccharide induces a monomer-dimer-tetramer assembly of FGF-2 while the disaccharide induces a monomer-dimer equilibrium. Evidence is presented indicating that the dimer conformation induced by the heparin octasaccharide is a side by side dimer with the FGF-2 molecules cis to the heparin, while the disaccharide-induced dimer is a head to head dimer in which FGF-2 molecules are trans to the ligand. These results, combined with previous studies, support the model that formation of a specific side by side heparin-induced FGF-2 dimer is required for activation of the FGF receptor.

Dimerization↗
PubMed1997

Modulation of fibroblast growth factor-2 receptor binding, dimerization, signaling, and angiogenic activity by a synthetic heparin-mimicking polyanionic compound.

Heparan sulfate (HS) proteoglycans play a key role in cell proliferation induced by basic fibroblast growth factor (FGF-2) and other heparin-binding growth factors. To modulate the involvement of HS, we have used a synthetic, nonsulfated polyanionic aromatic compound (RG-13577) that mimics functional features of heparin/HS. FGF-2-stimulated proliferation of vascular endothelial cells was markedly inhibited in the presence of 5-10 microg/ml compound RG-13577 (poly-4-hydroxyphenoxy acetic acid; Mr approximately 5 kD). Direct interaction between RG-13577 and FGF-2 was demonstrated by the ability of the former to compete with heparin on binding to FGF-2. RG-13577 inhibited FGF-2 binding to soluble- and cell surface-FGF receptor 1 (FGFR1). Unlike heparin, RG-13577 alone failed to mediate dimerization of FGF-2. Moreover, it abrogated heparin-mediated dimerization of FGF-2 and FGFR1, as well as FGF-2 mitogenic activity in HS-deficient F32 lymphoid cells. The antiproliferative effect of compound RG-13577 was associated with abrogation of FGF-2-induced tyrosine phosphorylation of FGFR1 and of cytoplasmic proteins involved in FGF-2 signal transduction, such as p90 and mitogen-activated protein kinase. A more effective inhibition of tyrosine phosphorylation was obtained after removal of the cell surface HS by heparinase. In contrast, tyrosine phosphorylation of an approximately 200-kD protein was stimulated by RG-13577, but not by heparin or FGF-2. RG-13577 prevented microvessel outgrowth from rat aortic rings embedded in a collagen gel. Development of nontoxic polyanionic compounds may provide an effective strategy to inhibit FGF-2-induced cell proliferation associated with angiogenesis, arteriosclerosis, and restenosis.

3T3 Cells↗
PubMed1997

Overlapping expression and redundant activation of mesenchymal fibroblast growth factor (FGF) receptors by alternatively spliced FGF-8 ligands.

FGF-8 is a member of the family of fibroblast growth factors and is expressed during vertebrate embryo development. Eight potential FGF-8 isoforms are generated by alternative splicing in mice, several of which are expressed during embryogenesis in epithelial locations. The significance of the multiple isoforms is currently unknown. In this report, we investigate the expression patterns and the specificity of the FGF-8 isoforms for known fibroblast growth factor (FGF) receptors. RNAs for seven of the eight potential isoforms are present at multiple sites of embryonic Fgf8 expression. None of the FGF-8 isoforms exhibited activity when assayed with BaF3 cells expressing the "b" splice forms of FGF receptors 1-3, which are mostly expressed in epithelial tissues. Mesenchymally expressed "c" splice forms of FGF receptors 2 and 3 and FGF receptor 4 were activated by several FGF-8 isoforms. These findings are consistent with the hypothesis that the multiple FGF-8 isoforms are functionally redundant and function to signal in paracrine (epithelial to mesenchymal) contexts.

Alternative Splicing↗
PubMed1996

The fibroblast growth factor receptor-1 is necessary for the induction of neurite outgrowth in PC12 cells by aFGF.

The PC12 subclone, fnr-PC12 cells, is defective in neurite outgrowth in response to acidic fibroblast growth factor (aFGF); however, its response to nerve growth factor (NGF) is normal. Examination of the expression of FGF receptors (FGFRs) revealed that although PC12 cells express FGFR-1, -3, and -4, fnr-PC12 cells have a reduced level of expression of FGFR-1 but not FGFR-3 and -4. Transfection of FGFR-1 into fnr-PC12 cells efficiently restored aFGF-induced neurite outgrowth, whereas transfection of FGFR-3 was much less efficient. Transfection of a chimeric receptor consisting of the extracellular domain of FGFR-3 fused to the transmembrane and intracellular domain of FGFR-1, termed FR31b, efficiently restored aFGF-induced neurite outgrowth. This demonstrates that the difference between these two receptors in their ability to induce neurite outgrowth is attributable to differences in the signaling capacity of their cytoplasmic domains. Activation of the chimeric receptor by aFGF induced a stronger and more persistent increase in the tyrosine phosphorylation of cellular proteins than did activation of FGFR-3 alone. In particular, the activation of MAP kinase by FR31b was more persistent than when activated by FGFR-3. This difference in signaling potential of FGFR-1 and -3 in fnr-PC12 cells may account for the difference in the potential for induction of neurite outgrowth. These results demonstrate that FGF-induced neurite outgrowth in PC12 cells occurs mainly via FGFR-1 and not via the other FGFRs expressed in these cells.

Amino Acid Sequence↗
PubMed1996

Receptor specificity of the fibroblast growth factor family.

Fibroblast growth factors (FGFs) are essential molecules for mammalian development. The nine known FGF ligands and the four signaling FGF receptors (and their alternatively spliced variants) are expressed in specific spatial and temporal patterns. The activity of this signaling pathway is regulated by ligand binding specificity, heparan sulfate proteoglycans, and the differential signaling capacity of individual FGF receptors. To determine potentially relevant ligand-receptor pairs we have engineered mitogenically responsive cell lines expressing the major splice variants of all the known FGF receptors. We have assayed the mitogenic activity of the nine known FGF ligands on these cell lines. These studies demonstrate that FGF 1 is the only FGF that can activate all FGF receptor splice variants. Using FGF 1 as an internal standard we have determined the relative activity of all the other members of the FGF family. These data should serve as a biochemical foundation for determining developmental, physiological, and pathophysiological processes that involve FGF signaling pathways.

Alternative Splicing↗
PubMed1996

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Apoproteins↗
PubMed1996

Expression and biological activity of mouse fibroblast growth factor-9.

Receptor specificity is an essential mechanism governing the activity of fibroblast growth factors (FGF). To begin to understand the developmental role of FGF-9/glial activating factor, we have cloned and sequenced the murine FGF-9 cDNA and expressed the protein in mammalian cells and in Escherichia coli. We demonstrate that the FGF-9 protein is highly conserved between mouse and human. Receptor specificity was determined by direct binding to soluble and cell surface forms of FGF receptor (FGFR) splice variants and by the mitogenic activity on cells, which express unique FGF receptor splice variants. Our data demonstrate that FGF-9 efficiently activates the "c" splice forms of FGFR2 and FGFR3, receptors expressed in potential target cells for FGF-9. Significantly, FGF-9 also binds to and activates the "b" splice form of FGFR3, thus becoming the first FGF ligand besides FGF-1 to activate this highly specific member of the FGF receptor family.

Alternative Splicing↗
PubMed1996

Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (Fgfr3) is a tyrosine kinase receptor expressed in developing bone, cochlea, brain and spinal cord. Achondroplasia, the most common genetic form of dwarfism, is caused by mutations in FGFR3. Here we show that mice homozygous for a targeted disruption of Fgfr3 exhibit skeletal and inner ear defects. Skeletal defects include kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae. Contrasts between the skeletal phenotype and achondroplasia suggest that activation of FGFR3 causes achondroplasia. Inner ear defects include failure of pillar cell differentiation and tunnel of Corti formation and result in profound deafness. Our results demonstrate that Fgfr3 is essential for normal endochondral ossification and inner ear development.

Achondroplasia↗
PubMed1996

Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia.

The longitudinal growth of the skeleton arises from the continuous process of endochondral ossification occurring at the ends of growing long bones. Dwarfism results when this process is disrupted, as in the autosomal dominant human skeletal diseases hypochondroplasia (HCH), achondroplasia (ACH) and thanatophoric dysplasia (TD). Interestingly, these disorders display a graded spectrum of phenotypic severity and are the result of distinct missense mutations in the fibroblast growth factor receptor 3 gene (FGFR3). TD, characterized by neonatal lethality and profound dwarfism, is the result of FGFR3 mutations, including an R248C substitution in the extracellular domain or a K650E substitution in the tyrosine kinase (TK) domain. ACH, which is non-lethal and presents less severe dwarfism, results almost exclusively from a G380R substitution in the transmembrane domain. Homozygous achondroplasia resembles the phenotype of TD. In this report the effect of the ACH and TD mutations on the activity and regulation of FGFR3 are analysed. We showed that each of the mutations constitutively activate the receptor, as evidenced by ligand-independent receptor tyrosine phosphorylation and cell proliferation. Moreover, the mutations that are responsible for TD were more strongly activating than the mutation causing ACH, providing a biochemical explanation for the observation that the phenotype of TD is more severe than that of ACH.

3T3 Cells↗
PubMed1995

FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides.

Fibroblast growth factors (FGFs) require a polysaccharide cofactor, heparin or heparan sulfate (HS), for receptor binding and activation. To probe the molecular mechanism by which heparin or HS (heparin/HS) activates FGF, small nonsulfated oligosaccharides found within heparin/HS were assayed for activity. These synthetic and isomerically pure compounds can activate the FGF signaling pathway. The crystal structures of complexes between FGF and these heparin/HS oligosaccharides reveal several binding sites on FGF and constrain possible mechanisms by which heparin/HS can activate the FGF receptor. These studies establish a framework for the molecular design of compounds capable of modulating FGF activity.

Animals↗
PubMed1995

FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development.

The Fgf8 gene is expressed in developing limb and craniofacial structures, regions known to be important for growth and patterning of the mouse embryo. Although Fgf8 is alternatively spliced to generate at least 7 secreted isoforms that differ only at their mature amino terminus, the biological significance of these multiple isoforms is not known. In this report, we demonstrate that multiple FGF-8 isoforms are present at sites of Fgf8 expression during mouse development. To address the possibility that the FGF-8 isoforms might interact with different fibroblast growth factor receptors, we prepared recombinant FGF-8 protein isoforms. We examined the ability of these proteins to activate alternatively spliced forms of fibroblast growth factor receptors 1-3, and fibroblast growth factor receptor 4. Recombinant FGF-8b and FGF-8c activate the 'c' splice form of FGFR3, and FGFR4, while FGF-8b also efficiently activates 'c' splice form of FGFR2. No activity could be detected for recombinant or cell expressed FGF-8a. Furthermore, none of the isoforms tested interact efficiently with 'b' splice forms of FGFR1-3, or the 'c' splice form of FGFR1. These results indicate that the FGF-8b and FGF-8c isoforms, produced by ectodermally derived epithelial cells, interact with mesenchymally expressed fibroblast growth factor receptors. FGF-8b and FGF-8c may therefore provide a mitogenic signal to the underlying mesenchyme during limb and craniofacial development.

Alternative Splicing↗
PubMed1995

BK1: an FGF-responsive central nervous system-derived cell line.

Fibroblast growth factors (FGF) are expressed at high levels in the central nervous system (CNS), however their function in the CNS is not well understood. The immortalized neuronal cell line (BK1), derived from a transgenic mouse central nervous system tumor, expresses high levels of FGF receptor 1 (FGFR1) and demonstrates both morphologic and biochemical changes when treated with basic FGF (FGF-2). We have derived subclones of BK1 cells with varying degrees of FGF responsiveness by transfecting either a wild type (FRW) or a truncated (FRX) form of FGFR1. Cells expressing high levels of FGFR1 rapidly and uniformly respond to FGF, while cells expressing FRX fail to respond to FGF, either morphologically or by the expression of molecular markers. These BK1 subclones will prove useful to study FGFR mediated signal transduction and FGFR responsive genes in a CNS derived cell. These studies also demonstrate that a dominant negative FGF receptor can be used as a tool to elucidate the function of FGF in the central nervous system.

Animals↗
PubMed1994

Murine FGFR-1 is required for early postimplantation growth and axial organization.

We have explored the role of fibroblast growth factor receptor 1 (FGFR-1) in early embryonic development using three experimental systems: genetically deficient mice, in vitro blastocyst culture, and FGFR-1-deficient embryonic stem cells. Using these systems, we demonstrate that FGFR-1 is required for proper embryonic cell proliferation and for the correct axial organization of early postimplantation embryos but not for mesoderm formation. FGFR-1-deficient embryos display severe growth retardation both in vitro and in vivo and die prior to or during gastrulation. Although these mutants can form nonaxial tissues, such as the allantois, amnion, and yolk sac mesoderm, they display defective patterning of the primitive streak and other axial structures, and frequently exhibit truncations or disorganization of posterior embryonic regions. Such abnormalities are unlikely to be caused by intrinsic blocks in mesodermal differentiation, as FGFR-1-deficient ES cell lines form teratomas consisting of many mesodermal cell types.

Animals↗
PubMed1994

Fibroblast growth factor receptor (FGFR) 3. Alternative splicing in immunoglobulin-like domain III creates a receptor highly specific for acidic FGF/FGF-1.

Fibroblast growth factors (FGF) regulate the growth and differentiation of cells through complex combinatorial signaling pathways. There are nine ligands that interact with a family of four tyrosine kinase FGF receptors (FGFR). Diversity in FGF signaling is determined in part by the affinity of specific ligand-receptor pairs. Alternative splicing in the FGFR ligand binding domain generates additional receptor isoforms with novel ligand affinities. For example, splicing events in the ligand binding domain of FGFR2 dramatically increases its affinity for keratinocyte growth factor (KGF/FGF-7). We have identified an alternatively spliced form of the FGFR3 mRNA, corresponding to known splice variants of FGFRs 1 and 2. We demonstrate both by binding studies on genetically engineered soluble receptors and by the mitogenic response of growth factor-dependent cell lines that this splice variant of FGFR3 (FGFR3 IIIb), by binding only acidic FGF (aFGF/FGF-1), has the most restricted ligand binding properties of any FGFR thus far described. Furthermore, by constructing a chimeric receptor that contains the homologous exon from FGFR2, we demonstrate that this single domain from FGFR2 is sufficient to confer upon FGFR3 the ability to bind KGF/FGF-7. The uniquely limited repertoire of ligands that interact with this receptor suggests that a novel ligand for FGFR3 IIIb exists.

Alternative Splicing↗
PubMed1994

Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morphologies.

Leukemia inhibitory factor (LIF) is a cytokine involved in embryonic and hematopoietic development. To investigate the effects of LIF on the lymphoid system, we generated a line of transgenic mice that expresses diffusible LIF protein specifically in T cells. These mice display two categories of phenotype that were not previously attributed to LIF overexpression. First, they display B cell hyperplasia, polyclonal hypergammaglobulinemia and mesangial proliferative glomerulonephritis, defects similar to those described for transgenic mice overexpressing the functionally related cytokine, interleukin-6. Secondly, the LIF transgenic mice display novel thymic and lymph node abnormalities. In the thymus, cortical CD4+CD8+ lymphocytes are lost, while numerous B cell follicles develop. Peripheral lymph nodes contain a vastly expanded CD4+CD8+ lymphocyte population. Furthermore, the thymic epithelium is profoundly disorganized, suggesting that disruption of stroma-lymphocyte interactions is responsible for many observed defects. Transplantation of transgenic bone marrow into wild type recipients transfers both the thymic and lymph node defects. However, transplantation of wild type marrow into transgenic recipients rescues the lymph node abnormality, but not the thymic defect, indicating the thymic epithelium is irreversibly altered. Our observations are consistent with a role for LIF in maintaining a functional thymic epithelium that will support proper T cell maturation.

Animals↗
PubMed1993

Benign prostatic hyperplasia in a transgenic mouse: a new hormonally sensitive investigatory model.

Recent advances in molecular biology have enabled incorporation of proto-oncogenes into the mouse germline. In this study we use a transgenic mouse line that overexpresses the fibroblastic growth factor (FGF) family member, int-2, under the control of mouse mammary tumor virus (MMTV) regulatory elements. One of the tissues targeted by MMTV is the mouse prostate. Expression of the MMTV-int-2 transgene in male transgenic mouse carriers results in a dramatic enlargement of the prostate gland which on histologic examination closely resembles the epithelial/glandular BPH observed in human and canine models. Pre- and postpubertal transgenic (NR) and wild-type (WT) FVB/N male mice were evaluated for the effects of hormonal manipulation by orchiectomy and orchiectomy followed by androgen replacement. Orchiectomy results in a significant decrease in size of the prostate in both NR and WT mice (p < 0.05), regardless of sexual maturity. Exogenous hormonal replacement with testosterone or dihydrotestosterone following orchiectomy results in significant regrowth of the prostate in both NR and WT mice. Flutamide, a potent nonsteroidal anti-androgen, resulted in a 55% reduction in size of the NR prostate (p < 0.002) and a similar 44% reduction in size of the WT prostate. Similarly, treatment of both NR and WT mice with leuprolide, a GnRH agonist, resulted in a significant decrease in prostate size (p < 0.05). Treatment of both NR and WT mice with finasteride (MK-906), a 5-alpha reductase inhibitor, failed to produce any significant regression in prostatic tissue. Based upon these data, we conclude that this transgenic mouse model, expressing int-2, produces an epithelial BPH histologically similar to other animal models. This transgenic model is hormonally sensitive and appears to represent a unique model for the investigation of BPH and growth factor induced epithelial cell hyperplasia.

5-alpha Reductase Inhibitors↗
PubMed1992

Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.

Amino Acid Sequence↗