Biomedical subjects

A Yayon

Publications and source records attributed to A Yayon.

At least 19 recordsLinked to original sources

PubMed2005

FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: implications for endochondral ossification.

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.

Achondroplasia↗

PubMed2002

FGF receptors ubiquitylation: dependence on tyrosine kinase activity and role in downregulation.

A crucial aspect of ligand-mediated receptor activation and shut-down is receptor internalization and degradation. Here we compared the ubiquitylation of either wild type or a K508A 'kinase-dead' mutant of fibroblast growth factor receptor 3 (FGFR3) with that of its naturally occurring overactive mutants, G380R as in achondroplasia, or K650E involved in thanatophoric dysplasia. Fibroblast growth factor receptors ubiquitylation was found to be directly proportional to their intrinsic tyrosine kinase activity, both of which could be blocked using kinase inhibitors. Despite excessive ubiquitylation, both overactive mutants failed to be efficiently degraded, even when challenged with ligand or overexpression of c-Cbl, a putative E3 ligase. We conclude that phosphorylation is essential for FGFR3 ubiquitylation, but is not sufficient to induce downregulation of its internalization resistant mutants.

Achondroplasia↗

PubMed2002

Mechanical resistance of biological repair cartilage: comparative in vivo tests of different surgical repair procedures.

The former common knowledge that cartilage lesions do not heal has been modified over the last few years due to new technologies. For repair of deeper circumscribed lesions osteochondral press-fit grafting and tissue engineering are used in clinical application. The histological data of the hyaline-like tissue obtained by engineering are just as satisfactory as the surviving grafted hyaline cartilage on top of osteochondral cylinders. But comparative studies are still lacking. To fill the gap and with a view to repairing larger osteoarthritic defects we have performed an in vivo study on 16 goats. Three months after the creation of a full thickness wide cartilage defect on the femoral condyle with harvesting of cartilage samples for tissue cultures we performed secondary cartilage repair procedures on the installed osteoarthritis areas: 1) grafting with autogenic osteochondral press-fit cylinders from the opposite knee, 2) autologous engineered chondrocyte grafting under periosteal flaps, 3) both in combination. The harvesting defects were either left as controls or filled with a hyaluronate fleece. After eight months the repaired areas and the harvesting defects were examined for cartilage stiffness as a novel comparative parameter. Compared to normal the cartilage on top of osteochondral grafts is considerably stiffer. Engineered cartilage is weaker than normal. Spontaneously ingrown fibrous cartilage is much weaker even with a carrier fleece. A combination of osteochondral press-fit grafts with engineered autologous cells restores biomechanical qualities to repaired larger degenerative cartilage defects.

Animals↗

PubMed2001

Mapping a heparin binding site on ErbB-3 epidermal growth factor receptor.

Signaling via the ErbB-family of receptors plays an important role in mammalian development and oncogenesis. Here we show that the ErbB-3 receptor, but not other members of this receptor family, binds to immobilized heparin and can be dissociated only at a high ionic strength comparable to that required for fibroblast growth factor receptors. Competition-binding analysis suggests that this interaction is specific and requires highly sulfated species of heparan sulfate. Primary sequence analysis of ErbB-3 identified a basic amino acid cluster (466)KHNRPRR(472) localized to the proximal, cysteine-rich extracellular ligand binding domain of the receptor, with charge density and distribution compatible with, but different to, known linear heparin binding motifs. Site-directed mutagenesis, replacing this sequence with the corresponding residues from ErbB-1, resulted in complete loss of heparin binding activity of the chimeric receptor. Finally, antibodies directed to the putative heparin binding peptide, efficiently bind the native receptor suggesting a novel target for blocking heparin mediated ErbB-3 interactions.

Alkaline Phosphatase↗

PubMed2001

Soluble and matrix-associated heparan sulfate proteoglycans increase expression of erb-B2 and erb-B3 in colon cancer cell lines.

We investigated the effect of hepatocyte-derived extracellular matrix (ECM) on the expression of erb-B2 and erb-B3 in colon cancer cell lines, as well as the role of erb-B2 and erb-B3 in colon cancer cell proliferation. Colon cancer cell lines plated on hepatocyte-derived ECM had increased protein levels of both erb-B2 and erb-B3. The addition of soluble recombinant proteoglycan syndecan-4 also resulted in higher expression of erb-B2 and erb-B3. We prepared hepatocyte-derived ECM from 1 to 7 days' cultures of hepatocytes, which contained different amounts of sulfated glycosaminoglycans. There was a direct correlation between the amounts of sulfated glycosaminoglycans in the ECM and the levels of erb-B2 and erb-B3 in the colon cell line KM12. The stimulatory effect of hepatocyte-derived ECM was abolished when the colon cancer cells were cultured in the presence of antibodies to erb-B2. These studies show that hepatocyte-derived ECM and the heparan sulfate proteoglycans present in it are responsible for inducing erb-B2 and erb-B3 in colon cancer cells. The growth stimulatory effect of extracellular matrix is mediated, at least in part, by increased expression of erb-B2 and erb-B3.

Antibodies, Monoclonal↗

PubMed2001

Basic fibroblast growth factor: a potential inhibitor of glutamine synthetase expression in injured neural tissue.

Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of glutamine synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Müller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of glutamine synthetase expression. This inhibition appears to be mediated by the c-Jun protein which accumulated, in response to bFGF, exclusively in Müller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c-Jun protein and a decline in glutamine synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down-regulates glutamine synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down-regulation of glutamine synthetase.

Animals↗

PubMed2001

Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces.

Fibroblast growth factors (FGFs) constitute a family of at least 20 structurally related heparin-binding polypeptides active in regulating cell growth, survival, differentiation and migration. FGF9, originally discovered as a glia-activating factor, shares 30% sequence identity with other FGFs and has a unique spectrum of target-cell specificity. FGF9 crystallized in the tetragonal space group I4(1), with unit-cell parameters a = b = 151.9, c = 117.2 A. The structure of the glycosylated protein has been refined to an R value of 21.0% with R(free) = 24.8%) at 2.6 A resolution. The four molecules in the asymmetric unit are arranged in two non-crystallographic dimers, with the dimer interface composed partly of residues from N- and C-terminal extensions from the FGF core structure. Most of the receptor-binding residues identified in FGF1- and FGF2-receptor complexes are buried in the dimer interface, with the beta8-beta9 loop stabilized in a particular conformation by an intramolecular hydrogen-bonding network. The potential heparin-binding sites are in a pattern distinct from FGF1 and FGF2. The carbohydrate moiety attached at Asn79 has no structural influence.

Amino Acid Sequence↗

PubMed2001

Development of a high throughput screening assay for inhibitors of fibroblast growth factor-receptor-heparin interactions.

High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.

Animals↗

PubMed2001

Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood.

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

Amino Acid Sequence↗

PubMed2001

Role of FGF9 and FGF receptor 3 in osteochondroma formation.

Osteochondromas are chondro-osseous protuberances that occur in metaphyses of long bones. The cartilaginous cap is assumed to be responsible for the growth of the lesions during childhood and adolescence, but mitotic figures are rarely seen in the cap. Therefore, another cell population, probably mesenchymal cells, is responsible for proliferation and growth. Residual mesenchymal cells capable of rapid proliferation are difficult to detect due to lack of specific histologic features. Two specific markers for mesenchymal cells, FGF receptor 3 (FGFR3) and collagen type IIa, have been described. Osteochondroma mesenchymal cells are found in the soft tissues overlying the cartilage cap. The surrounding areas of typical cartilage are negative for both mesenchymal cell associated antigens. The soft tissues overlying the cartilage do not have cartilaginous features. The undifferentiated cells overlying the exostosis yield in culture a rapidly proliferating homogenous population of fibroblast-like cells. Expression at the mRNA level of FGF9, FGFR3, and collagen type IIa is found in these cells, but not in skin fibroblasts from afflicted or healthy individuals. Exogenous administration of TGFbeta1 to cultures of hereditary multiple exostosis eliminates FGF9 expression. These results indicate fibrous regions contain the mesenchymal cells responsible for osteochondroma growth.

Bone Neoplasms↗

PubMed2000

Sorting polyclonal antibodies into functionally distinct fractions using peptide phage display: 'a library on top of a library'.

A general approach for sorting antibodies (Abs) to a restricted protein domain was developed using phage-displayed peptide libraries. The method is demonstrated by fractionating polyclonal antibodies (pAbs), raised against a short peptide derived from the extracellular, juxtamembrane region of fibroblast growth factor receptor 1 (FGFR1) into fractions with distinct chemical and biological characteristics. Screening two combinatorial peptide libraries, with the pAb, several sequences, homologous to different regions within the original peptide, were identified. Four of the corresponding peptides were synthesized and used as peptide-conjugated affinity columns for the fractionation of the pAbs. The fractions obtained were unique in their recognition patterns and in their capacity to immunoprecipitate and immunoblot, as well as to modulate the activity of FGFR1. This technique is, therefore, highly sufficient in separating pAbs to monospecific fractions and may also be used for fine mapping of different, even overlapping, sequences within a restricted peptide or protein domain.

Amino Acid Sequence↗

PubMed2000

Porphyrin analogues as novel antagonists of fibroblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis.

Fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) play a pivotal role in the multistep pathway of tumor progression, metastasis, and angiogenesis. We have identified a porphyrin analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra -p-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding and activation. TMPP demonstrated potent inhibition of binding of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at submicromolar concentrations. TMPP inhibits binding of radiolabeled FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an in vitro angiogenic assay measuring the extent of endothelial cell growth, tube formation, and sprouting, TMPP dramatically reduced the extent of the FGF2-induced endothelial cell outgrowth and differentiation. In a Lewis lung carcinoma model, mice receiving TMPP showed a marked inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon alternate daily injections of TMPP at 25 microg/g of body mass. Finally, novel meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel corrole-based derivative, with >50-fold increase in activity in vitro, had a significantly improved efficacy in blocking tumor progression and metastasis in vivo.

Animals↗

PubMed2000

Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice.

Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.

Animals↗

PubMed2000

Oligomerization reduces heparin affinity but enhances receptor binding of fibroblast growth factor 2.

The biological response of cells to fibroblast growth factors (FGFs) depends on heparan sulphate glycosaminoglycans sharing particular structural motifs. Heparin induced FGF dimerization has been suggested to mediate receptor dimerization and activation. Here we demonstrate that heparin-derived oligosaccharides that promote receptor binding and activation specifically induce the dimerization of basic FGF (FGF2). These heparin-induced dimers of FGF2 acquire high affinity for receptor binding and are biologically active. Using biotinylated FGF2 bound to immobilized streptavidin gradually saturated with biotin, enabled a quantitative analysis of heparin-dependent and heparin-independent FGF2 monomers and oligomers. Streptavidin induced FGF2 dimers bind and activate FGF receptors only in the presence of heparin. An excess of streptavidin, forcing biotin-FGF2 into monomers, reduces receptor binding and blocks FGF-dependent cell proliferation. All these suggest predominant receptor binding and activation by heparin associated FGF2 oligomers. Unexpectedly, heparin induced dimers and higher order oligomers lose most of their affinity towards heparin. Direct binding of soluble FGF receptors (FGFRs) to either monomers or dimers of FGF2, immobilized on heparin, confirm the preferred association of FGFRs with dimers of FGF2. Computerized molecular docking predicts a cis-oriented FGF2 dimer, stabilized by heparin, which complies with all the experimental data.

Animals↗

PubMed2000

Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization.

The crystal structure of a dimeric 2:2:2 FGF:FGFR:heparin ternary complex at 3 A resolution has been determined. Within each 1:1 FGF:FGFR complex, heparin makes numerous contacts with both FGF and FGFR, thereby augmenting FGF-FGFR binding. Heparin also interacts with FGFR in the adjoining 1:1 FGF:FGFR complex to promote FGFR dimerization. The 6-O-sulfate group of heparin plays a pivotal role in mediating both interactions. The unexpected stoichiometry of heparin binding in the structure led us to propose a revised model for FGFR dimerization. Biochemical data in support of this model are also presented. This model provides a structural basis for FGFR activation by small molecule heparin analogs and may facilitate the design of heparin mimetics capable of modulating FGF signaling.

Binding Sites↗

PubMed2000

The transmembrane mutation G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation.

A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation.

Amino Acid Substitution↗

PubMed1999

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.

Affinity Labels↗

PubMed1999

Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA.

Vascular endothelial growth factor (VEGF) is an important factor for endothelial cell proliferation and a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Its biological activity is mediated by two receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR). In contrast to VEGFR-2, a naturally occurring soluble form of the VEGFR-1 (sVEGFR-1) is produced by endothelial cells by differential splicing of the flt-1 gene, and it is a secreted gene product. In order to develop a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of sVEGFR-1, we established five anti-human receptor antibodies and characterized them in detail. These antibodies recognize different epitopes located within the seven Ig-like domains of the extracellular receptor protein but have no neutralizing activity in ligand binding assays. Together with a polyclonal antiserum, a specific human sVEGFR-1 ELISA was developed using the mAb #190.11. The ELISA can detect human sVEGFR-1 with a minimum detection limit of 1 ng/ml. The ELISA does not show any cross-reactivity with other related soluble receptors. Using this assay, human sVEGFR-1 was measured in the supernatant of different VEGFR-1 expressing cell types. No sVEGFR-1 protein was detectable after heparin Sepharose treatment or size-exclusion filtration (< 30 kDa). The ELISA assay for sVEGFR-1 was also used to measure the amount of the soluble receptor in amniotic fluid samples of patients undergoing amniocentesis during the course of normal pregnancies. The concentration of the samples was in the range of 5-35 ng/ml. This ELISA could be useful powerful tool for investigations concerning the physiological function of the soluble receptor under normal and pathophysiological conditions.Furthermore, it may facilitate studies of the mechanisms of receptor production.

Animals↗