Human dermal fibroblasts express multiple bFGF and aFGF proteins

Summary We investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions.     

Expression pattern of acidic and basic fibroblast growth factor genes in adult rat eyes

Although the retinal angiogenic and mitogenic factors have been identified to be acidic and basic fibroblast growth factors (aFGF and bFGF), little information has so far been available about the cells producing them and their function in retinal tissues. We found, by in situ hybridization, that the expression pattern of the aFGF gene differed remarkably from that of the bFGF gene in adult rat eyes. Our results demonstrated that the aFGF gene was produced by photoreceptor visual cells, neuronal cells in the inner nuclear layer and ganglion cells of the retina, in addition to pigment epithelial cells of the choroid, iris and ciliary body, and epithelial cells of the cornea, conjunctiva and lens, while bFGF was synthesized solely by the photoreceptor visual cells.     

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 phosphorylation and receptors in rod outer segments.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been isolated and purified from rod outer segments (ROS). aFGF is tightly bound to ROS membranes and can be specifically released by ATP. We show that this mechanism is dependent on the phosphorylation of aFGF itself. Phorbol 12-myristate 13-acetate (PMA) enhances this phenomenon independently of rhodopsin phosphorylation. This demonstrates that aFGF release from ROS membranes is dependent on its phosphorylation by endogenous kinase C. In addition specific binding sites for exogenous FGFs have been identified on ROS and disc membranes. A single high affinity site with a Kd of 40 pM was present in intact ROS while an additional low affinity site with a Kd of 300-600 pM was present in leaky ROS or in disc membranes. Light or ATP modified neither these Kd nor the apparent number of sites. The presence of specific receptors for FGFs and the kinase C dependent release of endogenous membrane bound aFGF suggest an autocrine mechanism which may be involved in photoreceptor cell biology.     

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

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

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

Background  

The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.     

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.     

Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: age dependency and brain region specificity.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are present in high levels in most areas of the embryonic rodent brain. To begin to understand the role of these growth factors in brain development, the effects of aFGF and bFGF on dissociated cell cultures prepared from embryonic and neonatal rat brain were studied. Addition of aFGF and heparin or bFGF alone to serum-free cultures of the dissociated Embryonic Day (E) 14.5 mesencephalon stimulates cell proliferation, as judged by [3H]thymidine autoradiography, leading to a maximal 75-fold increase in the total number of cells. This effect is dose-dependent with half-maximal increases at concentrations of about 5-6 ng/ml of aFGF or bFGF and is inhibited by the FGF antagonist HBGF-1U. The effect of aFGF on cell proliferation in cultures prepared from E14.5 mesencephalon is similar to that in cultures prepared from E14.5 cortex. However, in cultures prepared from E14.5 rhombencephalon or diencephalon, the proliferative effect of aFGF is much reduced. In all brain areas studied, the proliferative effect of aFGF declines with increasing age. Immunocytochemical analysis of E14.5 mesencephalic cultures demonstrated that the aFGF-induced increase in cell number is due to the proliferation of A2B5-immunoreactive (IR) glial precursor cells, but not of neuronal precursors, fibroblasts, or microglial cells. Moreover, differentiated glial fibrillary acidic protein-IR astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase-IR oligodendrocytes were not observed in cultures continuously treated with aFGF or bFGF, but were observed in high numbers after removal of the growth factors. These results suggest (1) that aFGF and bFGF are potent mitogens for glial precursor cells in all embryonic brain regions, (2) that the magnitude of the effects of aFGF depends on embryonic age and brain region, and (3) that both growth factors inhibit the differentiation of astrocyte or oligodendrocyte precursors. These observations made in vitro strongly support the hypothesis that FGF plays a critical role in gliogenesis and the timing of glial differentiation in the brain.     

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells

Summary The hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.     

Identification of a fibroblast growth factor-binding protein in Drosophila melanogaster.

As assessed by competitive binding and protein-crosslinking experiments, Drosophila melanogaster cells possess basic fibroblast growth factor (bFGF)-specific binding proteins that are similar to FGF receptors on vertebrate cells in molecular weight and binding affinity; these D. melanogaster cells, however, have no detectable binding proteins for acidic fibroblast growth factor (aFGF). Consistent with the presence of bFGF-specific binding proteins, D. melanogaster cells degrade bFGF but not aFGF. These results indicate the conservation of heparin-binding growth factors and receptors between vertebrates and D. melanogaster.     

The fibroblast growth factor family: Structural and biological properties

This article sumarizes the structural and biological properties of the family of fibroblast growth factors (FGF). Basic FGF (bFGF) and acidic FGF (aFGF) are the best characterized members of this family. bFGF and aFGF are potent modulators of cell proliferation, motility and differentiation. They are also potent angiogenesis factors in vivo. Some of the important biological characteristics of bFGF and aFGF discussed in the review include the affinity of bFGF and aFGF for heparin, their lack of secretion in culture and their association with extracellular matrix. Recently, several oncogenes, 40–50% homologous in sequence to bFGF and aFGF have been identified. These include int-2, hst, K-fgf and FGF-5. The structural and biological properties of these FGF-related oncogenes are also discussed.     

Signal transduction defect appears to be the cause of rat prostate cancer cell fibroblast growth factor insensitivity.

Using monolayer cultures of clonally isolated C3 and T5 rat prostate cancer cells, we determined that acidic (aFGF) and basic (bFGF) fibroblast growth factors profoundly enhanced T5 cell thymidine incorporation with half-maximum stimulation at 0.53 and 0.35 ng/ml, respectively. In contrast, aFGF or bFGF enhancement of C3 cell thymidine incorporation was about 5% of that of T5 cells, and effects were principally mitogen concentration independent. Saturation analyses and cross-linking studies established that both C3 and T5 cells contained high-affinity FGF receptors of 120 and 145 kilodaltons and that receptor content and Kd of C3 and T5 cells were comparable. aFGF or bFGF stimulation of T5 cell thymidine incorporation profoundly decreased as cell plating density was reduced from 1.5 x 10(5) to 1.0 x 10(4) cells/well. The modest response of C3 cells to either aFGF or bFGF also decreased as cell plating density was reduced. Because heparin preserves FGF biological activity and enhances bFGF binding to high-affinity FGF receptors, we examined the effect of heparin on FGF stimulation of C3 cell thymidine incorporation. We found that changes in cell plating density and/or medium heparin concentration had variable, inconsistent effects. These were C3 cell plating density associated and included inhibition or modest enhancement of FGF effects. Binding analyses established that high-affinity bFGF binding of C3 and T5 cells immediately prior to assessing FGF-stimulated thymidine incorporation was comparable and independent of cell plating density, implying that C3 cell FGF insensitivity was not attributable to differences in C3 and T5 cell FGF receptor content at the time of mitogen stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visual arrestin modulates gene expression in the retinal pigment epithelium: Implications for homeostasis in the retina

The retinal pigment epithelium (RPE) is essential for maintaining retinal homeostasis by removing and recycling photoreceptor outer segment (POS) in membranes. It also produces and secretes growth factors involved in retinal homeostasis. Arrestin 1 (ARR1) is specifically expressed in photoreceptors (PRs) and a vital molecule for keeping visual cycle between PRs and RPE. In the present study, we showed the expression of ARR1 was decreased by form-deprivation (FD) in retina of rat. The ARR1 was detected in the RPE of the controls but not in the RPE of FD, which indicates RPE phagocytes POS containing ARR1. Furthermore, we overexpressed ARR1 in cultured human RPE and revealed the ARR1 upregulates bFGF expression and downregulates TGF-β1, -β2 and bone morphogenetic protein-2 (BMP-2). The upregulation of bFGF by ARR1 directly works for PR survival and the downregulation of TGF-βs by ARR1 inhibits epithelial mesenchymal transition (EMT) of RPE, which is the underlying mechanism of keeping retinal homeostasis. Our results also indicate the regulation of ARR1 expression in RPE might become a novel therapeutic option for various ocular diseases.     

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.     

Cell fate decisions in a human retinal precursor cell line: basic fibroblast growth factor- and transforming growth factor-alpha-mediated differentiation

The purpose of this study was to determine if immortalized human retinal precursor cells could serve as a model to investigate cues that modulate cell fate and differentiation. We investigated the effects of a variety of growth factors broadly but specifically tested the effects of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)a in retinal cell differentiation and commitment. To determine the role of exogenously added growth factors in a human retinal precursor cell line (KGLDMSM), established from a first-trimester retina, cells were adapted to grow in a defined medium and exposed to a variety of trophic factors (epidermal growth factor [EGF], neuron growth factor [NGF], TGFalpha, TGFbeta, acidic FGF, and bFGF). Dose-response curves were developed to arrive at optimal concentrations. The neurotrophic potential of growth factors was determined by 3H-thymidine incorporation and bromodeoxyuridine (BrdU) labeling. The identity of the emerging neuronal phenotypes were determined by phase-contrast microscopy, immunolabeling for the neuron-specific antigens neurofilament protein (NF) and neuron-specific enolases (NSE), and photoreceptor-specific antigens (Rho1D4, 7G6) using immunocytochemistry and Western blot analysis. To identify some of the early response genes (c-fos, c-myc) expressed in response to growth factors, Northern blot analysis was performed. Almost all of the factors tested increased the total number of cells with a neuronal phenotype. Potency of growth factors to generate neurons was TGFalpha > bFGF > EGF > NGF. Both TGFalpha and bFGF, alone or in combination, increased the total number of neurons. Most of the neurons generated were photoreceptors, as depicted by the polarized phenotype, expression of photoreceptor-specific antigens, and processes resembling rudimentary outer segments. The increase in photoreceptor-like neurons is possibly attributable to an increase in numbers rather than greater survival. Additionally, the majority of the photoreceptors generated labeled with BrdU and for photoreceptor-specific antigens, suggesting that an inductive effect of bFGF and TGFalpha could occur in the cell cycle or shortly thereafter. Both bFGF and TGFalpha induced the expression of the early response gene c-fos while not altering the expression of c-actin or c-myc. The emergence of a photoreceptor phenotype was confirmed by both immunocytochemistry and Western blot analysis. The immortalized retinal precursor cell line could prove valuable in determining the role of exogenously added growth factors in retinal development and differentiation. Both bFGF and TGFalpha enhance the photoreceptor phenotype in medium-density cultures under conditions of defined medium. The same was confirmed by phase-contrast microscopy, immunocytochemistry, and Western blot analysis. Furthermore, cell fate determination in cultured precursor cells could occur during the late part of the cell cycle or shortly after completion of cell division. The effects of TGFalpha and bFGF seem to be slightly additive. The cell line will be extremely valuable in studying mechanisms of cell commitment and generation of retinal cell types, which could be tested for their potential for transplantation.