Presence Of FGF-2 Research Articles

Insulin and IGF-I Affect the Protein Composition of the Lens Fibre Cell with Possible Consequences for Cataract

Explanted newborn rat lens epithelial cells were cultured with various concentrations of FGF-2 and/or insulin or IGF-I for 8-20 days. The accumulation of alphaA-, alphaB-, betaA3/1-, betaB2- and gammaA-F-crystallin was measured. During culture with insulin only, i.e. in the absence of fibre cell differentiation, alphaA- and alphaB-crystallin accumulated to the same level as found in differentiating cells. Culture of epithelial cells with IGF-I led to an increase in alphaB-crystallin, but not in alphaA-crystallin. The addition of insulin under differentiation conditions (in the presence of 25 ng ml(-1)FGF-2) augmented the accumulation of alphaA-crystallin 1.5-fold, the accumulation of betaB2-crystallin two-fold and the accumulation of gammaA-F-crystallin five-fold over that found with FGF-2 only. The accumulation of alphaB- and betaA3/1-crystallin was not affected by insulin in the presence of FGF-2. Adding IGF-I to fibre cells differentiating in the presence of 25 ng ml(-1)FGF-2 resulted in a 1.5-fold increase (of questionable statistical significance) in both alphaA- and alphaB-crystallin and a two to three-fold increase in gammaA-F-crystallin compared to cells cultured with FGF-2 only, no significant effect of IGF-I on the accumulation of betaA3/1- or betaB2-crystallin was found. Comparison of the levels of mRNA and protein suggests that insulin acts to increase the level of transcription. Our results show that the response of fibre cells to insulin or IGF-I differs. Hence, even though half the maximum dosage required for the insulin effect was rather high (between 0.1 and >5 micro g), the effect of insulin cannot be merely transmitted by the IGF-I receptor. Our data further predict that insulin or IGF-I increases the overall ratio of beta- and gamma-crystallin to alpha-crystallin in the fibre cell, which could predispose the lens to cataract.

Identification of Neural Progenitors in the Adult Mammalian Eye

We have shown that the embryonic mammalian retina contains neural progenitors which display stem cell properties in vitro. Here we report the characterization of neural progenitors isolated from the adult mammalian eye. These quiescent cells, located in the pigmented ciliary bodies, proliferate in the presence of FGF2 and express the neuroectodermal marker nestin. The proliferating cells give rise to neural spheres and are multipotential; they express cell type-specific markers corresponding to neurons and glia. In addition, neural progenitors can generate secondary neural spheres, thus displaying potential to self-renew. The ciliary body-derived neural progenitors display retina-specific properties; the undifferentiated cells express Chx10, a retinal progenitor marker, and upon differentiation express markers corresponding to specific retinal cell types. Therefore, the pigmented ciliary body in the adult mammalian eye harbors neural progenitors that display stem cell properties and have the capacity to give rise to retinal neurons in vitro.

Dissection of the odontoblast differentiation process in vitro by a combination of FGF1, FGF2, and TGFbeta1.

Dental papillae (DP) isolated from first lower molars of 17-day-old mouse embryos were cultured in the presence of combinations of the following growth factors: FGF1, FGF2, and TGFbeta1. After 6 days in culture, only the DP treated with FGF1+TGFbeta1 contained differentiated odontoblast-like cells at the periphery of the explants, and these cells secreted extracellular matrix similar to predentin. Surprisingly, treatments with FGF2+TGFbeta1 induced cell polarization at the surface of the explants but no matrix secretion was observed. Electron microscopy and histochemical analysis of odontoblast markers showed that differentiated cells induced by FGF1+TGFbeta1 exhibited cytological features of functional odontoblasts with matrix vesicle secretion and mineral formation, positive alkaline-phosphatase activity, and type-I collagen production. DP cultured in the presence of FGF2+TGFbeta1 showed cell polarization and long and thin cell processes containing matrix vesicles; however, type-I collagen secretion was not detected and alkaline-phosphatase activity was completely inhibited. Our results indicate that, in our culture system, exogenous combinations of FGF1, FGF2, and TGFbeta1 interact with preodontoblasts and induce cell polarization or differentiation, which can be studied separately in vitro. Thus, FGF1 and TGFbeta1 do have a synergic effect to promote morphological and functional features of differentiated odontoblasts whereas FGF2 seems to modulate TGFbeta1 action, causing morphological polarization of preodontoblasts but limiting the functional activity of these cells in terms of type-I collagen secretion and alkaline-phosphatase activity.

FGF-2 Induces Surfactant Protein Gene Expression in Foetal Rat Lung Epithelial Cells Through a MAPK-independent Pathway

Fibroblast growth factors (FGFs) play important roles in diverse aspects of animal development including mammalian lung epithelial cell proliferation, differentiation, and branching morphogenesis. We developed an in vitro lung epithelial cell culture system to study functions and mechanisms of FGFs in regulating growth and differentiation of primary foetal rat lung epithelial cells. In comparison with other growth factors such as IGF-I, EGF, and HGF, FGFs were the most potent mitogens in stimulating lung epithelial cell proliferation. In the presence of FGF-1, 2, or 7, the primary lung epithelial cells could be propagated for generations and grown for more than two mo in vitro. Among the three FGFs tested, FGF-7 showed the strongest stimulation in cell growth. FGF-2, on the other hand, is the most effective inducer of lung epithelial cell-specific surfactant protein gene expression (SP-A, -B, and -C). FGF-2 upregulated SP-C expression in a dose-dependent manner. More interestingly, the induction of surfactant protein gene expression by FGF-2 appeared to be independent of MAPK pathway, since the SP-C expression was not inhibited but rather augmented by MEK1 inhibitor which inhibited MAPK activation and cell proliferation. Similar effects were observed for the expressions of surfactant protein genes SP-A and SP-B. In contrast to MAPK, FGF-2-induced SP-C expression was partially inhibited by PI 3-kinase inhibitor wortmannin. These data suggest dynamic roles and complex signalling mechanisms of FGFs in regulating lung epithelial cell proliferation and differentiation. While a MAPK-dependent pathway is essential for all three FGFs to stimulate cell proliferation, a MAPK-independent pathway may be responsible for the FGF-2-induced surfactant protein gene expression. PI 3-kinase may play an important role in mediating FGF-2-induced lung epithelial cell differentiation during development.

Monosomy 6 in human cultured fibroblast-like cells permanently stimulated by fibroblast growth factor 1: evidence for selection

The appearance of cells with monosomy 6 (mono6 cells) in cultures of human fibroblast-like cells during long-term stimulation with acidic fibroblast growth factor (FGF1) was confirmed in five of the six lines newly investigated. Aneugenic pretreatment at the start of the cultures accelerated the emergence of mono6 cells, as would be expected if selection, rather than induction, is the main mechanism involved. This could be confirmed by using an incidental rearrangement, der(8)t(6p;8p), that emerged in one of the lines by monitoring the proliferation of the mono6 cells (here monosomic for 6p22.1→qter) in mixtures with normal cells. During growth in the presence of FGF1, the proportion of mono6 cells increased six fold, whereas in the absence of FGF1, it declined to background levels. Selection rather than induction of the mono6 cells is further supported by their clonal origin, as ascertained on the basis of X-inactivation patterns in three informative cases. In addition, colonies grown in the presence of FGF1 from single cells did not reveal higher proportions of mono6 cells by fluorescence in situ hybridization analysis than those grown without the growth factor. During permanent stimulation with FGF1, the growth of mono6 cells did not become dependent on FGF1, nor did these cells lose their responsiveness to FGF1. Although evidence in favor of selection of preexistent mono6 cells by FGF1 is provided in this study, the contribution of a primary inducing mechanism cannot be entirely excluded.

Regulation of myogenesis by fibroblast growth factors requires beta-gamma subunits of pertussis toxin-sensitive G proteins.

Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Galpha or Gbeta gamma subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gbetagamma subunits are likely to be involved since the expression of the C terminus of beta-adrenergic receptor kinase 1, a Gbetagamma subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gbeta gamma dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gbeta gamma subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gbeta gamma subunits and activation of MAPKs to repress skeletal muscle differentiation.

Effects of FGFs on the morphogenic potency and AER-maintenance activity of cultured progress zone cells of chick limb bud.

The apical mesodermal region of chick limb buds (progress zone, PZ) which is essential for limb pattern formation contains uncommitted cells that change their positional values instructed by the apical ectodermal ridge (AER). Reciprically, the PZ cells maintain the AER activity. FGF-2 and FGF-4 can substitute for the AER to maintain normal outgrowth and gene expression in the limb bud. We examined the effects of FGF on the maintenance of PZ cells characteristics in culture by making recombinant limbs with anterior PZ cells that were pre-cultured in the presence of FGF-2 or FGF-4 and analyzed their morphogenic potency and responsiveness to positional cues arising from the zone of polarizing activity (ZPA). The limb buds expressed distal Hox genes and could form a segmented digit. Recombinant limb buds consisting of anterior PZ cells cultured without FGF failed to express Hox genes and formed instead a small cartilage nodule. These results indicate that addition of FGF-2 or FGF-4 to cultured PZ cells maintains their competence for Hox gene expression and digit formation, but not their responsiveness to positional cues from the ZPA. We also found that when anterior PZ cells which had been pre-cultured with FGF-2 or FGF-4 were implanted underneath the AER, they could maintain Fgf-8 expression in the AER, whereas this expression was not detected in the AER on the grafted anterior PZ cells that had been pre-cultured without FGF, indicating that FGF maintains AER-maintenance activity of PZ cells in culture.

The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity.

Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG "donor" cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR "reporter" cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 1) differentially regulates the binding and signaling of FGFs 1 and 2 and 2) acts as a positive regulator of FGF-2 signaling.

Lack of involvement of basic fibroblast growth factor (FGF-2) in capillary growth in skeletal muscles exposed to long-term contractile activity.

Fibroblast growth factors (FGFs) are involved in stimulation of angiogenesis in tumors and other pathological circumstances. Increased activity of normal skeletal muscles resulting from chronic electrical stimulation is a very potent stimulus for capillary growth but a relationship between the initiation of this angiogenesis and the involvement of autocrine growth factors has yet to be established. Although FGF expression has been reported in muscles stimulated for 3 weeks, capillary growth is underway significantly earlier, beginning around 3 days. The present experiments have therefore studied the possible involvement of basic fibroblast growth factor (FGF-2) in stimulated rat fast skeletal muscles prior to, and coincident with, capillary growth. Muscle contractions were induced via electrodes implanted in the vicinity of the peroneal nerve and maintained for 8h/day for 2, 4 or 7 days. Capillary/fiber ratio (C/F), based on staining of capillary endothelium for alkaline phosphatase, was not changed in either extensor digitorum longus (EDL) or tibialis anterior (TA) after 2 days stimulation, but increased in TA stimulated for 4 days and in both muscles after 7 days. The expression of mRNA for FGF-2, detected by ribonuclease protection assay, was decreased in all stimulated muscles compared with control or contralateral muscles; immunohistochemistry showed FGF-2 gene product in nerves and larger blood vessels but not in capillaries. There was no evidence from immunohistochemistry for up-regulation of receptors flg and bek for FGF-2. The presence of FGF-2, flg and bek in arterioles may indicate a possible role for FGF-2 in the regulation of blood flow since we have previously shown it to be a dilator of small arterioles. However, based on the lack of correlation between changes in capillary density and the expression of mRNA and protein for FGF-2 and its receptors, it is unlikely that it is directly linked with the initiation of angiogenesis resulting from chronic activity in skeletal muscles.

Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow.

We have investigated the effects of different growth factors on the proliferation and osteogenic potential of primary cultures of human bone marrow stromal cells (BMSC). Fibroblast growth factor (FGF)-2 was the most effective in promoting growth of these cells in vitro. The size of colonies formed in clonal conditions was approximately 2.5 times larger in presence of FGF-2. Also the morphology of BMSC was affected: cells cultured in 10% FCS alone became flattened, whereas FGF-2 expanded cells maintained a fibroblast-like elongated phenotype. Levels of alkaline phosphatase activity in BMSC expanded with FGF-2 were significantly lower (56%) than control and, after stimulation with ascorbic acid, betaGlycerophosphate and dexamethasone, FGF-2 expanded BMSC deposited approximately 3-fold more mineralized matrix than control cells. We have assessed osteogenicity of BMSC on hydroxyapatite porous scaffolds (bioceramics) by an ectopic bone formation assay. FGF-2 expanded BMSC yielded a higher bone formation (>20-fold) than control cells. We conclude that FGF-2, promoting BMSC proliferation, maintains cells in a more immature state allowing in vitro expansion of human osteo-progenitors which, associated with bioceramics, can differentiate in vivo and form bone tissue.

Cellular Proliferation and Fibroblast Growth Factors in the Corpus Luteum during Early Pregnancy in Ewes

To determine the relationship between cellular proliferation and the presence of FGF-1 and FGF-2 in the ovine corpus luteum (CL) during early pregnancy, ewes received an intravenous injection of bromodeoxyuridine (BrdU) 1 h before slaughter (n = 3/day) on day 12 after estrus (nonpregnant) or on days 12, 18, 24 or 30 after mating (pregnant). The labeling index (LI; number of BrdU-labeled nuclei expressed as a percentage of total nuclei) of each CL was determined by immunohistochemistry and subsequent image analysis. FGF-1 and FGF-2 were immunolocalized by using specific antibodies, and indirect immunoperoxidase detection. Moreover, FGF-2 was immunolocalized by using a primary antibody and fluorescein isothiocyanate (FITC)-labeled secondary antibody, and immunofluorescence was quantified by using an interactive laser cytometer and image analysis. Results demonstrated that the LI was similar for CL of non-pregnant and pregnant ewes on day 12 (4.27 ± 0.23 vs 5.10 ± 0.14%) and decreased (P < 0.05) from days 12–30 of pregnancy (2.73 ± 0.08, 2.02 ± 0.09 and 1.70 ± 0.04% on days 18, 24 and 30, respectively). FGF-1 was present in the cytoplasm of large and a few small parenchymal Meal cells, and the distribution and intensity of staining was similar for nonpregnant and pregnant ewes on day 12 as well as across days of pregnancy. In contrast, FGF-2 immunoreactivity was present only in luteal nonparen-chymal cells and interstitial areas and was greater (P < 0.05) for pregnant than nonpregnant CL on day 12 (2.34±0.12 vs 0.14±0.01%). Although FGF-2 immunoreactivity decreased (P<0.01) from days 12–30 of pregnancy (0.70±0.04, 0.22±0.01 and 0.06 ± 0.02% on days 18, 24, and 30, respectively), it was highly correlated (r = 0.99, P < 0.01) with luteal LI. We therefore suggest that FGF, and especially FGF-2, play a role in Meal cell proliferation or turnover during early pregnancy, and may thereby contribute to the maintenance of luteal function, which is critical for the successful establishment of pregnancy.

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.

Fibroblast Growth Factor-2 Can Mediate Cell Attachment by Linking Receptors and Heparan Sulfate Proteoglycans on Neighboring Cells

The myeloid 32D cell line, which grows in suspension and does not express FGF receptors or heparan sulfate proteoglycans, was transfected with the cDNA encoding FGF receptor-1 (32D-flg cells). When co-cultured with glutaraldehyde-fixed Chinese hamster ovary (CHO) cells, the 32D-flg cells remained in suspension in the absence of FGF-2 but attached to the CHO monolayer in the presence of 10 ng/ml FGF-2. In contrast, 32D cells transfected with the vector alone did not attach to the CHO monolayer in the presence of FGF-2. FGF-2-dependent attachment of 32D-flg cells was prevented by inclusion of 10 micrograms/ml heparin in the incubation medium and was diminished when CHO mutants in glycosaminoglycan synthesis or wild-type CHO cells treated with heparinase were used, indicating that the attachment occurred through FGF-2 interactions with heparan sulfates on the CHO cells. Attachment of 32D-flg cells to wild-type CHO cells was half-maximal at 0.4 ng/ml FGF-2 and was also observed with FGF-1 but not FGF-4. 32D-flg cells also attached to living CHO cells in a FGF-2-dependent manner, but attachment was transient at 37 degrees C. Induction of new proteins was not required for FGF-2-dependent attachment, since attachment occurred when the co-cultures were incubated at 4 degrees C and when the 32D-flg cells were preincubated with cycloheximide. FGF-2-dependent attachment of 32D-flg cells was also observed with Balb/C 3T3, NIH 3T3, and bovine capillary endothelial cells. We conclude that attachment is due to FGF-2 binding simultaneously to receptors on the 32D-flg cells and heparan sulfates on the CHO monolayers; thus, the FGF-2 acts as a bridge between receptor-expressing cells and heparan sulfate-bearing cells. In addition, induction of DNA synthesis in 32D-flg cells in response to FGF-2 was potentiated by the CHO-associated heparan sulfates to the same extent as by soluble heparin, indicating that this interaction has functional significance.

Human decidual leukocytes in first trimester normal pregnancy and pregnancy pathology

Human embryonic stem cells (hESC) differentiate into trophoblast when treated with BMP4. Here we studied the effects of either low (4% O2, L) or atmospheric O2 (20% O2, A) in the presence and absence of FGF2 on H1 hESC cultured in the presence of BMP4. Differentiation progressed from the periphery toward the center of colonies. It occurred most quickly in the absence of FGF2 and under A and was slowest in the presence of FGF2 and under L. Chorionic gonadotropin (CG) production required A, while FGF2 suppressed progesterone synthesis under both A and L. FGF2 was then omitted while we examined the trophoblast markers SSEA-1 and cytokeratin-7 and-8, whose expression also progressed inward from the periphery of colonies and occurred more rapidly under A than under L. By day 5, most cells outside central islands of Oct4-positive cells were positive for these antigens under both conditions and many also expressed HLA-G, a marker of extravillous cytotrophoblast. Under A, but not L, CGα and CGβ became prominent in GATA2-positive, peripherally located, multinucleated cells. In conclusion, BMP4 induced conversion of hESC exclusively toward trophoblast; FGF2 slowed differentiation, while O2 accelerated this process and promoted syncytiotrophoblast formation.

Fibroblast growth factor (FGF)-2 sense and antisense mRNA and FGF receptor type 1 mRNA are present in the embryonic and adult rat nervous system: specific detection by nuclease protection assay

In the present study, we attempted to clarify the controversial question whether basic fibroblast growth factor (bFGF, FGF-2) mRNA is present or absent in the embryonic central nervous system (CNS). For this purpose we analyzed the expression of the FGF-2 mRNA in the embryonic and adult forebrain, brainstem, and spinal cord using the highly specific ribonuclease protection assay. Using this method we were able to detect FGF-2 mRNA in the rat CNS of embryonic day (E) 16 and 17, however, at lower levels compared to adult FGF-2 mRNA levels. In addition, we show that a FGF-2 antisense transcript is expressed in embryonic CNS tissue. Furthermore, using this method, we demonstrate FGF receptor 1 mRNA in the rat embryonic and adult CNS. The presence of FGF-2 and FGF receptor 1 suggests a physiological role for this growth factor during the development of the embryonic CNS.

Models of in Vitro Angiogenesis: Endothelial Cell Differentiation on Fibrin But Not Matrigel Is Transcriptionally Dependent

Endothelial cells can be induced to form a branching network of tubular structures using a variety of cell culture conditions. We have examined this differentiation process using several sets of conditions: plating human umbilical vein endothelial cells (HUVEC) on Matrigel, adding collagen to the apical surface of HUVEC grown on fibronectin, and plating HUVEC on fibrin in the presence of FGF-1. We determined that although the first two conditions produce dramatic morphologic changes in the HUVEC population, gene transcription and translation are not required for the regulation of the process. Rather, post-translational events are involved since the Matrigel-dependent process could be inhibited by the addition of nocodazole, suramin or H7, a protein kinase inhibitor. In contrast, the fibrin matrix-dependent differentiation pathway involved transcriptional and translational events since the addition of either actinomycin D or cycloheximide inhibited this pathway. A modified differential display of RNA extracted from HUVEC after 0, 2, 5, and 24 hours on fibrin revealed expression of a novel cDNA.

High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2.

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.

Cell Cycle-Dependent Nuclear Localization of Exogenously Added Fibroblast Growth Factor-1 in BALB/c 3T3 and Human Vascular Endothelial Cells

Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.

Bumetanide and furosemide inhibited vascular endothelial cell proliferation.

In this study, we examined the role of the bumetanide-sensitive Na+/K+/Cl-cotransport in the mitogenic signal of vascular endothelial cell proliferation. The activity of the Na+/K+/Cl- cotransport is dramatically decreased in quiescent subconfluent cells, as compared to subconfluent cells growing in the presence of FGF. The Na+/K+/Cl- cotransport activity of quiescent subconfluent cultures deprived of FGF decreased to 6%, whereas that of quiescent cells grown to confluency was reduced to only 33% of the activity of subconfluent cells growing in the presence of FGF. The basal low activity of Na+/K+/Cl- cotransport in the quiescent subconfluent vascular endothelial cells was dramatically stimulated by FGF. In order to explore the role of the Na+/K+/Cl- cotransport in the mitogenic signal of the endothelial cells, the effect of two specific inhibitors of the cotransport -furosemide and -bumetanide was tested on cell proliferation induced by FGF. Bumetanide and furosemide inhibited synchronized cell proliferation measured by direct counting of cells and by DNA synthesis. Inhibition by furosemide and bumetanide was reversible; removal of these compounds completely released the cells to proliferate. These results indicate that the effect of these drugs is specific and is not due to an indirect toxic effect. This study clearly demonstrates that the FGF-induced activation of the Na+/K+/Cl- cotransport plays a role in the mitogenic signal pathway of vascular endothelial cells.

Fibroblast growth factors enhance dopamine fiber formation from nigral grafts

Members of the fibroblast growth factor (FGF) family have earlier been shown to exert potent trophic effects on cells of both the central and peripheral nervous system. The presence of FGF-1 and -2 (FGF-1, acidic FGF; FGF-2, basic FGF) has recently been demonstrated in the dopaminergic cells of substantia nigra in rat and FGF-2 has been shown to be able to increase survival and promote neurite outgrowth of cultured mesencephalic neurons. In the presence study, we have investigated possible trophic effects of FGF-1 and FGF-2 on developing rat ventral mesencephalon of different fetal stages by utilizing the in vivo method of intraocular transplantation to sympathetically denervated hosts. Survival and growth of developing grafts after growth factor treatment was followed in oculo. The Falck-Hillarp technique was used for evaluation of catecholaminergic fiber outgrowth into the host iris in whole-mount preparations. FGF-2 significantly increased the volume of the mesencephalic grafts when compared to grafts treated with the vehicle alone. The mean volume of FGF-1-treated grafts was larger than that of control grafts, but this difference was not statistically significant. FGF-1 significantly increased the area of outgrowth of dopaminergic fibers into the host iris without a corresponding increase in the number of dopaminergic neurons, as evaluated by TH immunohistochemistry. FGF-2 had no effect on dopaminergic fiber outgrowth on grafted E14 ventral mesencephalon but it did have a significant effect on fiber outgrowth from E15 and E16 grafts. Moreover, the FGF-2 treated E16 grafts contained a larger number of dopaminergic neurons as compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)