Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action

The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ectodermal cells has recently been shown to regulate limb morphogenesis via presentation of fibroblast growth factor (FGF) 4/FGF 8 while expression on tumor cells was shown to sequester hepatocyte growth factor and promote tumor dissemination. To date, however, CD44 HSPG expression in tissue macrophages and lymphocytes has not been adequately investigated, despite the fact these cells actively synthesize growth factors and chemokines and indirect evidence that monocyte CD44 sequesters macrophage inflammatory protein-1beta. Here we show primary human monocytes rather than lymphocytes express CD44 HSPGs, but only following in vitro differentiation to macrophages or activation with the proinflammatory cytokine interleukin-1alpha or bacterial lipopolysaccharide. Furthermore, we show these isoforms are preferentially modified with heparan rather than chondroitin sulfate, bind the macrophage-derived growth factors FGF-2, vascular endothelial growth factor, and heparin-binding epidermal growth factor with varying affinities (K(d) 25-330 nM) and in the case of FGF-2, can stimulate productive binding to the high affinity tyrosine kinase FGF receptor 1 (FGFR1). In contrast, we find no evidence for significant binding to C-C chemokines. Last, we confirm by immunofluorescent antibody staining that inflamed synovial membrane macrophages express CD44 HSPGs and that expression is greatest in cells containing high FGF-2 levels. These results suggest a paracrine role for macrophage CD44 HSPG isoforms in the regulation of growth factor action during inflammation.     

Fibroblast and epidermal growth factors modulate proliferation and neural cell adhesion molecule expression in epithelial cells derived from the adult mouse tongue

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.     

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.     

Characterization of FGF receptor expression in human neutrophils and their contribution to chemotaxis

Several members of the fibroblast growth factor (FGF) family are potent endothelial cell (EC) mitogens and angiogenic factors, and their activities can be mediated by four tyrosine kinase receptors (FGFR1-4). In addition, FGFs can induce the release of inflammatory mediators by ECs and the expression of adhesion molecules at their surface, thereby favoring the recruitment and transvascular migration of inflammatory cells such as neutrophils. Neither the expression nor the biological activities that could be mediated by FGFRs have been investigated in human neutrophils. By biochemical and cytological analyses, we observed that purified circulating human neutrophils from healthy individuals expressed varying levels of FGFRs in their cytosol and at their cytoplasmic membrane. FGFR-2 was identified as the sole cell surface receptor, with FGFR-1 and -4 localizing in the cytosol and FGFR-3 being undetectable. We assessed the capacity of FGF-1 and FGF-2 to induce neutrophil chemotaxis in a modified Boyden microchamber and observed that they increase neutrophil transmigration at 10(-10) and 10(-9) M and by 1.77- and 2.34-fold, respectively, as compared with PBS-treated cells. Treatment with a selective anti-FGFR-2 antibody reduced FGF-1-mediated chemotaxis by 75% and abrogated the effect of FGF-2, while the blockade of FGFR-1 and -4 partially inhibited (15-40%) FGF-chemotactic activities. In summary, our data are the first to report the expression of FGF receptors in human neutrophils, with FGF-1 and FGF-2 promoting neutrophil chemotaxis mainly through FGFR-2 activation.     

Cellular characterization of epidermal growth factor-expanded free-floating neurospheres.

Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, alpha- and beta-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.     

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.

Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions.     

Properties of a fetal multipotent neural stem cell (NEP cell)

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFR alpha), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.     

Normal Human Fibroblasts Produce Membrane-Bound and Soluble Isoforms of FGFR-1

Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which αFGF and βFGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.     

Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors.

Ligand-induced dimerization and transphosphorylation are thought to be important events by which receptor tyrosine kinases generate cellular signals. We have investigated the ability of signalling-defective, truncated fibroblast growth factor (FGF) receptors (FGFR-1 and FGFR-2) to block the FGF response in cells that express both types of endogenous FGF receptors. When these dominant negative receptors are expressed in NIH 3T3 cells transformed by the secreted FGF-4, the transformed properties of the cells can be reverted to various degrees, with better reversion phenotype correlating with higher levels of truncated receptor expression. Furthermore, truncated FGFR-2 is significantly more efficient at producing reversion than FGFR-1, indicating that FGF-4 preferentially utilizes the FGFR-2 signalling pathway. NIH 3T3 clones expressing these truncated receptors are more resistant to FGF-induced mitogenesis and also exhibit reduced tyrosine phosphorylation upon treatment with FGF. The block in FGF-signalling, however, can be overcome by the addition of excess growth factor. The truncated receptors have binding affinities that are four- to eightfold lower than those of wild-type receptors, as measured by Scatchard analysis. We also observed a partial specificity in the responses of truncated-receptor-expressing clones to FGF-2 or FGF-4. Our results suggest that the block to signal transduction produced by kinase-negative FGF receptors is achieved through a combination of dominant negative effects and competition for growth factor binding with functional receptors.     

Alternatively spliced FGFR-1 isoforms differentially modulate endothelial cell activation of c-YES

Ligand activation of fibroblast growth factor receptor-1 (FGFR-1) induces an angiogenic response following activation of multiple intracellular signaling substrates, including the Src family of nonreceptor tyrosine kinases (SFK). However, the direct association between FGFR-1 and SFK and the involvement of SFK in FGFR-1-dependent cell proliferation have been controversial. Structural variants of FGFR-1 are generated by alternative splicing which results in two major isoforms, containing either three (FGFR-1α) or two (FGFR-1β) immunoglobulin-like domains in the extracellular region. To determine whether alternatively spliced FGFR-1 isoforms differentially activate SFK, we have examined FGF receptor-negative endothelial cells stably transfected with human cDNA encoding either FGFR-1α or FGFR-1β. Transient activation of c-YES, the predominant SFK expressed in these endothelial cells, was restricted to FGFR-1β transfectants following exposure to acidic fibroblast growth factor (FGF-1). Co-immunoprecipitation studies revealed that c-YES directly associated with FGFR-1β. The Src homology (SH)2 domain (and not the SH3 domain) of c-YES was able to recognize tyrosine phosphorylated FGFR-1β. FGFR-1β-specific activation of c-YES was accompanied by its association with and activation of cortactin. FGF-1 treatment of both FGFR-1α and FGFR-1β transfectants induced SFK-independent cellular proliferation and growth in low density cultures. At high density, under both anchorage-dependent and -independent conditions, FGF-1 failed to induce proliferation and growth of FGFR-1α transfectants. In contrast, FGF-1 induced proliferation, growth, and formation of cord-like structures in high density cultures of FGFR-1β transfectants in an SFK-dependent manner. In vitro cord formation on Matrigel was restricted to FGFR-1β transfectants in an SFK-dependent manner. Formation of vascular structures in vivo was limited to endothelial cells transfected with FGFR-1β. Collectively, these results emphasize the roles of alternatively spliced FGFR-1 structural isoforms and activation of SFK as modulators of endothelial cell growth during the formation of neovascular structures.     

Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF- 2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2- treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.     

c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts

Activated fibroblast growth factor receptor 1 (FGFR1) propagates FGF signals through multiple intracellular pathways via intermediates FRS2, PLCgamma, and Ras. Conflicting reports exist concerning the interaction between FGFR1 and Src family kinases. To address the role of c-Src in FGFR1 signaling, we compared proliferative responses of murine embryonic fibroblasts (MEF) deficient in c-Src, Yes, and Fyn to MEF expressing either endogenous levels or overexpressing c-Src. MEF with endogenous c-Src had significantly greater FGF-induced DNA synthesis and proliferation than cells lacking or overexpressing c-Src. This was related directly to c-Src expression by analysis of c-Src-deficient cells transfected with and sorted for varying levels of a c-Src expression vector. This suggests an "optimal" quantity of c-Src expression for FGF-induced proliferation. To determine if this was a general phenomenon for growth factor signaling pathways utilizing c-Src, responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and lysophosphatidic acid (LPA) were examined. As for FGF, responses to EGF were clearly inhibited when c-Src was absent or overexpressed. In contrast, varying levels of c-Src had little effect on responses to PDGF or LPA. The data show that mitogenic pathways activated by FGF-1 and EGF are regulated by c-Src protein levels and appear to differ significantly from those activated by PDGF and LPA.     

Ligand binding properties of binary complexes of heparin and immunoglobulin-like modules of FGF receptor 2

Epithelial cells, which express FGFR2IIIb, bind and respond to FGF-1, FGF-7 and FGF-10, but not FGF-2. Stromal cells, which bind and respond to FGF-1 and FGF-2, but not FGF-7 and FGF-10, express FGFR2IIIc or FGFR1IIIc. Here we show that when both isolated FGFR2betaIIIb and FGFR2betaIIIc or their common Ig module II are allowed to affinity select heparin from a mixture, the resultant binary complexes bound FGF-1, FGF-2, and FGF-7 with nearly equal affinity. In addition, FGF-2 and FGF-7 bound to both heparin-Ig module IIIb and IIIc complexes, but FGF-1 bound to neither Ig module III. The results show that in isolation both Ig modules II and III of FGFR2 can interact with heparin and that each exhibits a binding site for FGF. We suggest that the specificity of FGFR2IIIb and FGFR2IIIc is dependent on the cell membrane environment and heparin/heparan sulfate. Ig modules II and III cooperate both within monomers and across dimers with cellular heparan sulfates to confer cell type-dependent specificity of the FGFR complex for FGF.     

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.     

Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture

One characteristic of skeletal muscle differentiation is the conversion of proliferating cells to a population that is irreversibly postmitotic. This developmental change can be induced in vitro by depriving the cultures of specific mitogens such as fibroblast growth factor (FGF). Analysis of cell surface FGF receptor (FGFR) in several adult mouse muscle cell lines and epidermal growth factor receptor (EGFR) in mouse MM14 cells reveals a correlation between receptor loss and the acquisition of a postmitotic phenotype. Quiescent MM14 cells, mitogen-depleted, differentiation-defective MM14 cells, and differentiated BC3H1 muscle cells (a line that fails to become postmitotic upon differentiation) retained their cell surface FGFR. These results indicate that FGFR loss is not associated with either reversible cessation of muscle cell proliferation or biochemical differentiation and thus, further support a correlation between receptor loss and acquisition of a postmitotic phenotype. Comparison of the kinetics for growth factor receptor loss and for commitment of MM14 cells to a postmitotic phenotype reveals that FGFR rises transiently from approximately 700 receptors/cell to a maximum of approximately 2,000 receptors/cell 12 h after FGF removal, when at the same time, greater than 95% of the cells are postmitotic. FGFR levels then decline to undetectable levels by 24 h after FGF removal. During the interval in which FGFR increases and then disappears there is no change in its affinity for FGF. The transient increase in growth factor receptors appears to be due to a decrease in ligand-mediated internalization because EGFR, which undergoes an immediate decline when cultures are deprived of FGF (Lim, R. W., and S. D. Hauschka. 1984. J. Cell Biol. 98:739-747), exhibits a similar transient rise when cultures are grown in media containing both EGF and FGF before switching the cells to media without these added factors. These results indicate that the loss of certain growth factor receptors is a specific phenotype acquired during skeletal muscle differentiation, but they do not resolve whether regulation of FGFR number is causal for initiation of the postmitotic phenotype. A general model is presented in the discussion.