Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7)

A newly identified member of the fibroblast growth factor (FGF) family, designated FGF-10, is expressed during development and preferentially in adult lung. The predicted FGF-10 protein is most related to keratinocyte growth factor (KGF, or FGF-7). The latter is unique among FGFs in that it binds and signals only through the FGF receptor (FGFR2b) isoform KGF receptor (KGFR) expressed specifically by epithelial cells. In order to examine the biological and biochemical properties of human FGF-10, we isolated the cDNA and expressed its encoded protein in bacteria. The recombinant protein (rFGF-10) was a potent mitogen for Balb/MK mouse epidermal keratinocytes with activity detectable at 0.1 nM and maximal at around 5 nM. Within this concentration range, FGF-10 did not stimulate DNA synthesis in NIH/3T3 mouse fibroblasts. rFGF-10 bound the KGFR with high affinity comparable to that of KGF, and did not bind detectably to either the FGFR1c (Flg) or FGFR2c (Bek) receptor isoforms. The mitogenic activity of FGF-10 could be distinguished from that of KGF by its different sensitivity to heparin and lack of neutralization by a KGF monoclonal antibody. These results indicate that FGF-10 and KGF have similar receptor binding properties and target cell specificities, but are differentially regulated by components of the extracellular matrix.     

A functional fibroblast growth factor-1 immunoglobulin fusion protein

Proteins of the fibroblast growth factor (FGF) family play diverse roles in embryonic development, angiogenesis, and wound healing. The most well studied targets of FGF activity typically are cells of mesodermal and neuroectodermal origin; in addition, expression of FGF-1 (acidic FGF) is increased at several sites of chronic immunologic injury, and recent studies show that FGF-1 also may interact with cells of the immune system. In some human T cells, FGF-1 can induce signals necessary for production of interleukin-2, a key cytokine required for T cell proliferation. To better characterize the interaction of FGF-1 with FGF receptors on T cells, a fusion protein was constructed containing a portion of the constant region of human IgG1 (Fc) at the amino terminus of FGF-1. The Fc-FGF-1 fusion protein retained FGF function as determined by stimulation of tyrosine phosphorylation and DNA synthesis in NIH 3T3 cells. Binding of the intact fusion protein to FGF receptor 1 (FGFR1) on T cells was demonstrated by immunoprecipitation of the receptor bound to Fc-FGF-1 and by flow cytometry showing binding of fusion protein to T cells expressing FGFR1. This functional Fc-FGF-1 protein should prove useful in identifying FGFR-expressing cells.     

The glycine box: a determinant of specificity for fibroblast growth factor

Acidic fibroblast growth factor (FGF-1), keratinocyte growth factor (FGF-7), and FGF-10 are homologues with distinct specificity. In the presence of heparin, FGF-1 binds and activates in vitro all FGFR subtypes, while FGF-7 exhibits absolute specificity for the IIIb splice variant of FGFR2. FGF-10 exhibits a similar specificity but also binds the FGFR1IIIb isoform. Neither FGF-7 nor FGF-10 will bind to IIIc isoforms of FGFR. Molecular models of FGF, heparin, and the FGFR ectodomain suggested that sequences between beta-strands 10 and 12 of FGF may be important for the interaction of FGF with the heparin-FGFR ectodomain duplex. Site-directed mutants of FGF-7 and FGF-10 were prepared to test whether this domain might underlie failure of FGF-7 and FGF-10 to bind to the FGFRIIIc isoforms. Constructions with substitution of FGF-1 sequences spanning the entire C-terminus encoded in exon 3 or only C-terminal sequences spanning beta-strands 10 through 12 conferred ability on FGF-7 to bind to and activate FGFRIIIc without a significant loss in binding to or activation of FGFR2IIIb. A series of twelve different substitutions of shorter segments of FGF-1 sequences into the C-terminal portion of FGF-7 or FGF-10 revealed that substitution of GSCKRG for GIPVRG or the tri-peptide sequence KKN for NQK just N-terminal to it conferred dual activities on both the FGF-7 and FGF-10 backbones. The results suggest that the combined sequence domain, which we call the FGF glycine box (G-box), is a major determinant for the specificity of the binding of FGF to heparan sulfate-FGFR duplexes.     

Fibroblast growth factor 2 promotes pancreatic epithelial cell proliferation via functional fibroblast growth factor receptors during embryonic life

Several investigators have postulated that soluble growth factors are involved in the early development of the pancreas. In many tissues in which soluble factors are implicated in development, these factors act on their target cells through tyrosine kinase receptors. Because we had some preliminary evidence that fibroblast growth factor receptors (FGFRs) were expressed in the early pancreas, we investigated the effect of fibroblast growth factors (FGFs) during embryonic pancreatic development. For that purpose, we first studied the distribution and the functionality of FGFRs during pancreatic organogenesis. FGFR1 and FGFR4 were shown to be expressed at a high level during early pancreatic development before embryonic day 16, their levels of expression decreasing thereafter. The functionality of FGFR was studied next. It was demonstrated in vitro that both FGF1 and FGF2 induce the expression of NGFI-A mRNA, a useful indicator of functional growth factor-signaling pathways. Finally, the effect of FGF2 on embryonic pancreatic epithelial cell proliferation was studied. It was shown that FGF2 induces the proliferation of pancreatic epithelial cells during embryonic life. Taken together, these data strongly suggest that FGFs are implicated in pancreatic development during embryonic life.     

Genome instability in BVDV: an examination of the sequence and structural influences on RNA recombination

To determine the extent to which autocrine effects of acidic fibroblast growth factor (FGF)-1 overexpression contribute to an increased malignant phenotype, FGF-1-transfected MCF-7 cells were retransfected with a FGF receptor (FGFR1) vector encoding a truncated dominant-negative receptor to inhibit autocrine FGF signal transduction. This transfection eliminated FGF signaling within the breast cancer cells without interfering with their ability to produce FGF-1, thereby allowing possible paracrine effects to still be observed in vivo. Truncated FGFR1 overexpression inhibited the acquired ability of FGF-1-overexpressing cells to form colonies in soft agar in estrogen-depleted or antiestrogen-containing medium. However, soft agar colony formation was still stimulated by estrogen treatment in cells expressing up to 6 x 10(5) truncated FGFR1 sites per cell. In vivo, truncated receptor expression severely inhibited the ability of the FGF-1-overexpressing cells to form tumors without estrogen in ovariectomized mice, indicating that the mitogenic effect of FGF-1 on the breast tumor cells was important in the estrogen-independent in vivo growth of these transfectants. However, rapid formation of large tumors was still observed in estrogen-supplemented mice injected with the truncated FGFR1-expressing cells, suggesting that the paracrine effects of FGF production could act in synergy with mitogenic effects mediated by estrogen. Truncated FGFR1-overexpressing cells also continued to form tumors in tamoxifen-treated mice, raising the possibility that the paracrine effects of FGF-1 expression may allow the partial agonist properties of this antiestrogen to be more readily observed. We conclude that autocrine effects of FGF-1 increase the ability of MCF-7 breast cancer cells to grow in vitro and in vivo under estrogen-depleted conditions but that paracrine effects of FGF-1 are also involved in the enhancement of tumor growth in estrogen-supplemented or tamoxifen-treated animals.     

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.     

Modulation of fibroblast growth factor receptor expression and signalling during retinoic acid-induced differentiation of Tera-2 teratocarcinoma cells

We have analyzed the regulation of fibroblast growth factor receptors (FGFRs) during retinoic acid (RA) induced differentiation of Tera-2 human embryonal carcinoma cells. Undifferentiated Tera-2 cells expressed mRNAs for all four known FGFRs. Their differentiation led to loss of FGFR-4 mRNA expression and mRNA levels for FGFR-2 and FGFR-3 were considerably downregulated, whereas the mRNA levels for FGFR-1 remained unaltered. A substantial decrease in binding of K-FGF was found to occur upon RA-induced differentiation of the cells. In undifferentiated Tera-2 cells FGF stimulation caused an increase of c-fos mRNA, and c-jun mRNAs, but no increase of junB mRNA, whereas in the differentiated cells, FGFs strongly stimulated the expression of all three genes. Thus differentiation of the Tera-2 cells leads to marked changes in FGFR gene expression as well as to complex alterations in their responses to exogenous FGFs.     

Inhibition of growth of malignant rat prostate tumor cells by restoration of fibroblast growth factor receptor 2

A loss of expression of fibroblast growth factor (FGF) receptor 2 IIIb (FGFR2IIIb), which responds to stroma-derived FGF, accompanies progression of premalignant androgen-responsive rat prostate tumor epithelial cells to the malignant phenotype. Concurrently, the level of FGFR2 gene expression is reduced and lost altogether in over 30% of cells, whereas all malignant cells abnormally express FGFR1, which is normally confined to stromal cells (S. Feng et al., Cancer Res., 57:5369-5378, 1997). To determine the relative roles of the FGFR2 and FGFR1 kinases in growth of malignant cells, we transfected malignant prostate epithelial cells with the wild-type FGFR2IIIb kinase and an artificial chimeric construct (FGFR2IIIb/R1) composed of the FGFR2IIIb ectodomain and the FGFR1 kinase domain. Population growth kinetics, in both the absence and presence of FGF-7, which binds only the FGFR2IIIb ectodomain, were then examined in the transfected cell populations. In contrast to the untransfected malignant tumor cells and those expressing the FGFR2IIIb/R1 chimera, FGF-7 caused a dose-dependent net inhibition of the population growth rates of cells expressing the full-length FGFR2IIIb kinase. The results suggest that although the FGFR2 kinase can mediate positive mitogenic effects, it mediates a net restriction on the growth of prostate tumor epithelial cells relative to FGFR1. Highly malignant prostate tumor cells, which have lost the FGFR2 tyrosine kinase, retain the cellular response mechanisms to it. Restoration of the FGFR2 kinase to malignant tumors that are refractory to treatment may present a new avenue for gene therapy.     

Characterization of bovine serum factor triggering the lysis of liposomes via complement activation

Heparin-binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin-binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR1/flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF-2 during EAE and determined the cellular origin of FGF-2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF-2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF-2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed colocalization of FGF-2 to activated microglia/macrophages with strongest expression in the macrophage-rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/macrophage in an autocrine fashion, to induce the expression of other factors like insulin-like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/or regeneration of oligodendrocytes within the lesion area.     

Itraconazole oral solution versus clotrimazole troches for the treatment of oropharyngeal candidiasis in immunocompromised patients

The development of the nervous system appears to be under the control of multiple growth factors, neurotrophins and cytokines, which may be expressed either continuously or transiently throughout defined stages of cellular generation, proliferation or differentiation. Fibroblast growth factor (FGF) cytokines and their receptors are abundantly expressed in the embryonic nervous system but their localization at autonomic levels in the fetal spinal cord has not yet been detailed. Immunoreactivity to FGF-2, probably the best characterized member of the FGF family (FGF-1 to FGF-10) and of one of its high affinity receptors, FGFR-1, was found in autonomic neurons at embryonic day E14, the peak day of generation and proliferation in the common ventral motoneuron pool. It was also continuously present throughout the investigated subsequent stages (E15 to postnatal day P30). Immunogold electron microscopy revealed the cytoplasmic localization of FGF-2 and FGFR-1 in intermediolateral neurons, the major group of sympathetic preganglionic neurons in the spinal cord. In these neurons, immunocytochemistry from E14 onwards showed the co-distribution of both markers at the period of axonal outgrowth to peripheral targets, e.g. the adrenal medulla. Our findings suggest autocrine and/or paracrine actions of FGF-2 for sympathetic preganglionic development but do not support its role as a target-derived neurotrophic factor for autonomic neuron development.     

Common and specific determinants for fibroblast growth factors in the ectodomain of the receptor kinase complex

The assembly and activation of oligomeric complexes of FGF, the transmembrane receptor kinase (FGFR), and heparan sulfate transmit intracellular signals regulating growth and function of cells. An understanding of the structural relationships between the three subunits and their redundancy and specificity is essential for understanding the ubiquitous FGF signaling system in health and disease. Previously, we reported that a primary heparin or heparan sulfate binding site resides in a distinct sequence in immunoglobulin (Ig)-like module II of the three modules of FGFR. Here we report that in the absence of flanking sequences, isolated Ig module II of FGFR1 supports the binding of FGF-1, FGF-2, and FGF-7 in respective order of affinity. None of the three FGFs detectably bind Ig module I or the IIIb and IIIc splice variants of Ig module III in the absence of flanking sequences. Ig module I and the C-terminus of Ig module III are dispensable for high-affinity binding of FGF-1, FGF-2, and FGF-7. Alterations in highly conserved Ig module II in the heparin binding domain and substitution of individual sequence domains spanning the entire sequence of Ig module II with those from Ig module I obliterated FGF binding. Addition of a specific number of FGFR sequences to the C-terminus of Ig module II resulted in a gain in affinity for FGF-7. Several site-specific alterations in the C-terminus of full-length FGFR1IIIc, an isoform that otherwise absolutely rejects FGF-7, resulted in gain of FGF-7 binding. These results suggest that a complex of Ig module II and heparan sulfate is the base common active core of the FGFR ectodomain and that flanking structural domains modify FGF affinity and determine specificity.     

Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression

Fibroblast growth factor 7 (FGF-7) or keratinocyte growth factor (KGF), is a potent and specific mitogen for epithelial cells. We have recently identified a novel human FGF-7 homologue, named FGF-10. To study the expression of this new FGF family member and its regulation in wound repair, we cloned the mouse FGF-10 (mFGF-10) cDNA. The encoded protein is 92% identical to human FGF-10 and 91% identical to rat FGF-10. When expressed in mammalian 293 cells, the mFGF-10 protein was glycosylated but remained cell- or extracellular matrix-associated. Upon addition of heparin, mFGF-10 protein was released into the media. mRNA encoding mFGF-10 was relatively abundant in lung, skin, brain and heart. In the skin, both FGF-7 and mFGF-10 were expressed in the dermal, but not the epidermal compartment. In contrast to FGF-7, mFGF-10 expression was not induced during cutaneous wound repair. In cultured fibroblasts, expression of mFGF-10 was strongly repressed by transforming growth factor beta and tumor necrosis factor alpha, whereas epidermal growth factor and interleukin-1beta had no effect. These results demonstrate a differential regulation of mFGF-10 and FGF-7 expression in vitro and during the wound healing process.     

Expression of fetal kidney growth factors in a kidney tumor line: role of FGF2 in kidney development

To identify growth factors which may play a role in kidney organogenesis, we have analyzed culture supernatants from the pediatric kidney tumor cell line G401. G401 cells were found to secrete fibroblast growth factor 2 (FGF2), a potent mitogen for mesenchymal cells, OP-1/BMP7, an epithelial cell growth inhibitor, and midkine (MK). Northern blotting confirmed expression of FGF2, OP-1/BMP7 and MK mRNA, as well as Wnt5A mRNA in G401 cells. In situ hybridization and immunocytochemistry on human fetal kidney demonstrated FGF2 expression in epithelial cells of the branching ureteric bud epithelium, nephron precursors ("S-shaped bodies"), proximal tubule epithelium and the parietal epithelium of the glomerulus. FGF2 protein in condensed "caps" of induced mesenchymal cells was also detected by immunocytochemistry. FGF2 protein was found to be concentrated in nuclei, particularly in proximal tubule epithelial cells. Recombinant FGF2 was found to act as a mitogen on primary mouse fetal kidney cell cultures. The results demonstrate G401 cells secrete a variety of fetal kidney growth factors and that FGF2 may act as a mitogen for fetal kidney cells and thus could play a role in the morphogenesis of the kidney.     

Identification of the cytoplasmic regions of fibroblast growth factor (FGF) receptor 1 which play important roles in induction of neurite outgrowth in PC12 cells by FGF-1

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.     

Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization

Platelet factor 4 (PF-4) inhibits angiogenesis in vitro and in vivo. The mechanism of inhibition is poorly understood. We have investigated the mechanism of inhibition by examining the interaction of PF-4 and the fibroblast growth factor-2 (FGF-2)/fibroblast growth factor receptor (FGFR) system. PF-4 inhibited the binding of FGF-2 to high-affinity and low-affinity binding sites in murine microvascular endothelial cells (LEII cells) and proliferation. Maximum inhibition of binding to endothelial FGF receptors was observed at PF-4 concentrations between 5 and 10 microg/mL (half maximum inhibition at 0.6 micro/mL), and proliferation was completely inhibited at 2 microg/mL. At this concentration, PF-4 reduced internalization of 125I-FGF-2 by threefold and delayed degradation. To gain insight into the mechanism of inhibition, we have analyzed the interaction of PF-4 with FGF-2/FGFR by using mutant heparan sulfate-deficient Chinese hamster ovary (CHO) cells transfected with the FGFR-1 cDNA (CHOm-FGFR-1) and by examining the direct interaction with FGF-2. In the absence of heparin, PF-4 inhibited binding of 125I-FGF-2 to CHOm-FGFR-1 cells in a concentration-dependent manner, although not completely. In the presence of heparin, PF-4 abolished totally the stimulatory effect of heparin. Furthermore, PF-4 complexed to FGF-2 and inhibited endogenous or heparin-induced FGF-2 dimerization. These results indicate that PF-4 interacts with FGF-2 by complex formation, inhibiting FGF-2 dimerization, binding to FGF receptors, and internalization. This mechanism most likely contributes to the antiangiogenic properties of PF-4.     

Growth factor regulation of cell growth and proliferation in the nervous system. A new intracrine nuclear mechanism

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crestderived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.     

Molecular cloning, nucleotide sequence, and function of a site-specific recombinase encoded in the major ''pathogenicity island'' of Salmonella typhi

Fibroblast growth factor (FGF)-10 is a novel member of the FGF family. Although FGF-10 mRNA was preferentially expressed in the lung, the mRNA was also expressed, although at low levels, in the brain. We examined the localization of FGF-10 mRNA along with FGF-7 mRNA in the rat brain by in situ hybridization. FGF-10 mRNA showed spatially restricted expression in some regions of the brain, including the hippocampus, thalamus, midbrain and brainstem, although FGF-7 mRNA was not expressed in any of the brain regions examined. FGF-10 mRNA was strongly expressed in several restricted nuclei, especially in motor nuclei, including the oculomotor nucleus, dorsal motor nucleus of vagus, motor trigeminal nucleus, facial nucleus and hypoglossal nucleus. This localization pattern was distinct from those of aFGF, bFGF FGF-5 and FGF-9 mRNAs reported previously. The cellular localization of FGF-10 mRNA showed that the mRNA in the brain was preferentially expressed in neurons but not in glial cells. The present findings indicate that FGF-10, an additional member of the FGF family expressed in the brain, has a distinct role in the brain.     

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [125I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [125I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4 degrees C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-beta 1. Scatchard analysis revealed the presence of approximately 1.0 x 10(5) FGF-2 binding sites per cell with an apparent Kd of 1.2 x 10(-10) M. Interestingly, the binding of [125I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.     

Amphibian FGF-1 is structurally and functionally similar to but antigenically distinguishable from its mammalian counterpart

Recent studies have shown that fibroblast growth factors (FGF) play an important role in the diverse cellular mechanisms involved with vertebrate development. One system which has received a great deal of attention is the developing limb in part because of the extensive epithelial-mesenchymal interactions that take place during this process. Because it closely parallels the developmental process of the limb and is a model for wound repair, the phenomenon of amphibian limb regeneration has been used to investigate the role of FGF in these processes. We have recently reported on the cloning and functional characterization of an FGF receptor (FGFR) isolated from amphibian regenerative tissue. In this report, we describe the isolation and characterization of an FGF-1 molecule from the newt, Notophthalmus viridescens. Amino acid sequence comparisons indicate that the newt FGF-1 exhibits between 79 to 83% identity with FGF-1 from mammalian and avian species. The full length cDNA of the newt FGF-1 was cloned into a prokaryotic expression vector and purified from E. coli. Although the newt FGF-1 shares a high degree of primary amino acid sequence similarity with other FGF-1 molecules, the recombinant protein was not detected in a Western blot analysis using a polyclonal antibody directed against mammalian FGF-1. Despite the antigenic divergence, the newt FGF-1 was capable of binding to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGFRs with dissociation constants comparable to those reported for mammalian FGF-1. Newt FGF-1 could also be cross-linked to receptors on the surface of NIH/3T3 cells. In addition, it elicits a mitogenic response in NIH/3T3 cells indistinguishable from human recombinant FGF-1.