Fibroblast growth factor receptor signaling in Xenopus retinal axon extension

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin–stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin–stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca²⁺/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin– and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 633–641, 1998     

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

Fibroblast growth factors (FGFs) are a family of heparin-binding growth factors. FGFs exert their pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. Their activity is modulated by a variety of free and extracellular matrix-associated molecules. Also, the cross-talk among FGFs, vascular endothelial growth factors (VEGFs), and inflammatory cytokines/chemokines may play a role in the modulation of blood vessel growth in different pathological conditions, including cancer. Indeed, several experimental evidences point to a role for FGFs in tumor growth and angiogenesis. This review will focus on the relevance of the FGF/FGF receptor system in adult angiogenesis and its contribution to tumor vascularization.     

Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats

Background

Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet.

Methodology/Principal Findings

Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch''s membrane of ZD-LE rats varied between 0.4–3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch''s membrane or even inside it.

Conclusions/Significance

In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch''s membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch''s membrane.     

Effects of TGF-beta on retinal pigmented epithelium in vitro

Embryonic chick retinal pigmented epithelium (RPE) has been grown on glass derivatized with covalently bound proteins of basement membrane and treated with transforming growth factor-beta (TGF-beta). In the present paper we show that over the concentration range tested (0.1-10 ng/ml) TGF-beta has no effect on RPE cell proliferation either in the presence or the absence of serum, cell motility and the organization of cytoskeleton-extracellular matrix linkage complexes with respect to their structure and presence of actin, vinculin, talin, integrin and fibronectin. The protein profiles of total cell/ECM extracts of cells grown in the presence or the absence of TGF-beta are similar although some stimulation of protein synthesis and of production of fibronectin-containing extracellular matrix has been detected.     

Fibroblast growth factor receptor levels decrease during chick embryogenesis

Two putative receptors for fibroblast growth factor (FGF) of approximately 150 and 200 kD were identified in membrane preparations from chick embryos. Specific binding (femtomoles/milligram) of 125I-aFGF to whole chick embryonic membranes was relatively constant from day 2 to 7, then decreased fivefold between days 7 and 13. Day-19 chick embryos retained 125I-aFGF binding at low levels to brain, eye, and liver tissues but not to skeletal muscle or cardiac tissues. The 200-kD FGF receptor began to decline between day 4.5 and 7 and was barely detectable by day 9, whereas the 150-kD FGF receptor began to decline by day 7 but was still detectable in day-9 embryonic membranes. It is not known whether the two FGF-binding proteins represent altered forms of one polypeptide, but it is clear that their levels undergo differential changes during development. Because endogenous chick FGF may remain bound to FGF receptor in membrane preparations, membranes were treated with acidic (pH 4.0) buffers to release bound FGF; such treatment did not affect 125I-aFGF binding and moderately increased the number of binding sites in day-7 and -19 embryos. Consequently, the observed loss of high affinity 125I-aFGF binding sites and FGF-binding polypeptides most likely represents a loss of FGF receptor protein. These experiments provide in vivo evidence to support the hypothesis that regulation of FGF receptor levels may function as a mechanism for controlling FGF-dependent processes during embryonic development.     

Melanosome metabolism in the retinal pigmented epithelium of the opossum

Summary Melanosomal metabolism, including both formation and degradation of melanosomes, was studied in the retinal pigmented epithelium (RPE) of the adult opossum. The majority of the observations were made on a transitional zone between the tapetal and non-tapetal RPE, the region where melanosome metabolism was at its highest level. Formation of melanosomes, demonstrated ultrastructurally by the presence of stage-II and -III premelanosomes, was also examined autoradiographically following the incorporation of the melanin precursor, dihydroxyphenylalanine. The autoradiographic evidence indicated that many newly formed melanosomes were rapidly incorporated into complexes. Ultrastructural observations suggested that melanosome complexes were formed by at least two methods, via the fusion of melanosomes with phagosomes derived from outer segments of photoreceptors, or by the sequestration of melanosomes by cisternae. A central finding of this study, supported by both ultrastructural and histochemical data, is that there are specialized cellular regions that vary in melanosomal formation and lysosomal activity. Stage-II premelanosomes were observed only in the basal parts of the RPE cells, whereas stage-III and -IV melanosomes were found primarily in the apical RPE. Both ultrastructural and cytochemical observations indicated that degradation of melanosomes occurs only in the basal RPE. These findings are interpreted in terms of the expression of both tapetal and nontapetal characteristics in transitional cells. Finally, this study illustrates the role of lysosomal enzymes in shaping the pattern of pigmentation, and shows that the association of lysosomal activity with melanosomes depends on the functional state of the melanosome.This investigation was supported by National Institutes of Health research grant EY 01429 and, in part, by a Bob Hope award from Fight for Sight, Inc., New York City (to R.H. Steinberg), and a Fight for Sight, Inc. Summer Fellowship to K.G. Herman     

Characterization of gingival epithelium epidermal growth factor receptor

The binding characteristics of gingival epithelium epidermal growth factor (EGF) receptor were investigated using epithelial cell membranes from bovine gingiva. The binding of [125I]EGF was found to be time and protein concentration dependent, reversible, and specific. Unlabeled EGF competed for [125I]EGF binding with IC50 of 0.25nM and maximum displacement of 93% at 0.81nM. Scatchard analysis of the binding data inferred the presence of two binding sites, one of high affinity (Kd = 3.3 nM and Bmax = 47.3fmol/mg protein) and the other of a low affinity (Kd = 1.6 microM and Bmax = 1.9pmol/mg protein). Crosslinking of [125I]EGF to gingival membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed a receptor protein of 170kDa.     

Proteomic analysis of mature melanosomes from the retinal pigmented epithelium

The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.     

Fibroblast growth factor receptor modulators employing diamines with reduced phospholipidosis-inducing potential

SUN13837 (1), a fibroblast growth factor receptor modulator, has been an attractive candidate for treating neurodegenerative diseases. However, one of its metabolites, N-benzyl-4-(methylamino)piperidine (BMP), turned out to possess phospholipidosis-inducing potential (PLIP) in vitro. To obtain SUN13837 analogs with reduced phospholipidosis risk, we replaced BMP with other diamines possessing low PLIP. Our effort led to the discovery of compound 6 with increased efficacy. Further structural modifications to reduce hydrogen bond donors afforded 17 with improved brain exposure. Oral administration of 17 at 1 mg/kg once daily for 10 days showed enhanced recovery of coordinated movement in a rat acute stroke model, suggesting that it is a promising follow-up compound for 1 with reduced risk of phospholipidosis.     

Fibroblast growth factor signaling in tumorigenesis

Fibroblast growth factors and their signaling receptors have been associated with multiple biological activities, including proliferation, differentiation and motility. Consequently, they have evoked interest as candidate oncogenes with the potential to initiate and/or promote tumorigenesis. This has resulted in a large literature describing the presence of these growth factors and their receptors in cancer cell lines and primary tumors of diverse origin. However, it is only recently that compelling evidence has emerged to implicate the fibroblast growth factors (Fgfs) and their receptors in the genesis of human cancers. Here, we outline the model systems that demonstrate the potential oncogenic nature of Fgf signaling and summarise recent evidence that implicates aberrant Fgf signaling as important in the natural history of some common human cancers.     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.     

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.     

Components of the cytoskeleton in the retinal pigmented epithelium of the chick

The retinal pigmented epithelium (RPE) is a simple cuboidal epithelium with apical processes which, unlike many epithelia, do not extend freely into a lumen but rather interdigitate closely with the outer segments of the neural retina. To determine whether this close association was reflected in the cytoskeletal organization of the RPE, we studied the components of the cytoskeleton of the RPE and their localization in the body of the cell and in the apical processes. By relative mobility on SDS gels and by immunoblotting, we identified actin, vimentin, myosin, spectrin (240/235), and alpha-actinin as major components, and vinculin as a minor component. In addition, the RPE cytoskeleton contains polypeptides of Mr 280,000 and 250,000; the latter co-electrophoreses with actin-binding protein. By immunofluorescence, the terminal web region appeared similar to the comparable region of the intestinal epithelium that consists of broad belts of microfilaments containing myosin, actin, spectrin, and alpha-actinin. However, the components of the apical processes were very different from those of intestinal microvilli. We observed staining along the process for myosin, actin, spectrin, alpha-actinin, and vinculin. The presence in the apical processes of contractile proteins and also of proteins typically found at sites of cell attachments suggests that the RPE may actively adhere to, and exert tension on, the neural retina.