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PubMed1998

The expression and localization of fibroblast growth factor-1 (FGF-1) and FGF receptor-1 (FGFR-1) in human breast cancer.

Fibroblast growth factor-1 (FGF-1) is an inducer of angiogenesis, the growth of new blood vessels. The expression and localization of FGF-1 (acidic FGF) and FGF receptor (FGFR)-1 in mammary tissues from patients with breast cancer was investigated using Western blot analysis and immunohistochemistry. The affinity-purified FGF-1 antibody which did not have cross-reactivity to FGF-2 (basic FGF) was used in this study. Western blot analysis demonstrated the presence of FGF-1 protein in all of the samples from breast cancer, but not benign tumors such as mastopathy and fibroadenoma. To assess the localization of FGF-1 in cancer tissues, immunostaining with specific antibody was performed. All samples from breast cancer displayed significantly intense staining with FGF-1 antibody. The extent and intensity of immunoreactive FGF-1 polypeptides in cancer cells was statistically much greater than those of cells from fibroadenoma or mastopathy. Control immunostaining with normal rabbit serum or anti-FGF-1 antibody adsorbed with the recombinant FGF-1 polypeptide was completely negative. In contrast to FGF-1, Western blot analysis demonstrated the presence of FGFR-1 protein in all of the samples from breast cancer and benign tumors. By immunohistochemical analysis, the enhanced expression of FGFR-1 was observed in breast cancer cells. Benign tumor cells or interstitial cells displayed a faint expression of FGFR-1. These results demonstrated that breast cancer cells not only generated FGF-1, but also expressed FGFR-1, and FGF-1 might play a role in the proliferation of breast cancer cells not only by paracrine but also by autocrine mechanism.

Adult↗
PubMed1988

Clonal analysis of vertebrate myogenesis. VIII. Fibroblasts growth factor (FGF)-dependent and FGF-independent muscle colony types during chick wing development.

The effect of bovine fibroblast growth factor (FGF) on the in vitro differentiation of various stage-specific populations of skeletal muscle colony-forming (MCF) cells from the developing chick wing bud was examined. The results show that bovine FGF (3 ng/ml daily) delays the onset of differentiation of MCF cells obtained from Day 4-12 wing buds by about 1 day; but, in addition, the results demonstrate that a subset of colony-forming cells derived from stage 23-27 (Day 4-5) embryos require FGF for myogenic differentiation. The FGF-dependent MCF cells attach and grow in the absence of FGF, but do not differentiate unless given FGF within 1-3 days after inoculation. Thus, between stages 23 and 27 the myogenic population contains discrete subclasses that are FGF dependent and others that are FGF independent. Both subclasses are found within two of the previously classified MCF cell populations, the early and late MCF cells. FGF-dependent and independent early MCF cells are present within the wing bud until stage 25, after which only the FGF-independent early MCF subclass persists. Similarly, both FGF-dependent and -independent late MCF cells are present between stages 25 and 27, but only the FGF-independent late MCF subclass remains after stage 31. The mechanisms responsible for relative changes in the proportions of MCF cell subclasses and for the FGF requirements are not understood. In addition, while FGF is required, there is no evidence suggesting that FGF triggers skeletal muscle terminal differentiation within the FGF-dependent MCF cell subclasses.

Animals↗
PubMed2005

Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display.

Fibroblast growth factor-binding proteins (FGF-BP) are secreted carrier proteins that release fibroblast growth factors (FGFs) from the extracellular matrix storage and thus enhance FGF activity. Here we have mapped the interaction domain between human FGF-BP1 and FGF-2. For this, we generated T7 phage display libraries of N-terminally and C-terminally truncated FGF-BP1 fragments that were then panned against immobilized FGF-2. From this panning, a C-terminal fragment of FGF-BP1 (amino acids 193-234) was identified as the minimum binding domain for FGF. As a recombinant protein, this C-terminal fragment binds to FGF-2 and enhances FGF-2-induced signaling in NIH-3T3 fibroblasts and GM7373 endothelial cells, as well as mitogenesis and chemotaxis of NIH-3T3 cells. The FGF interaction domain in FGF-BP1 is distinct from the heparin-binding domain (amino acids 110-143), and homology modeling supports the notion of a distinct domain in the C terminus that is conserved across different species. This domain also contains conserved positioning of cysteine residues with the Cys-214/Cys-222 positions in the human protein predicted to participate in disulfide bridge formation. Phage display of a C214A mutation of FGF-BP1 reduced binding to FGF-2, indicating the functional significance of this disulfide bond. We concluded that the FGF interaction domain is contained in the C terminus of FGF-BP1.

Amino Acid Sequence↗
PubMed1995

Androgen and fibroblast growth factor (FGF) regulation of FGF receptors in S115 mouse mammary tumor cells.

We studied the androgen regulation of fibroblast growth factor (FGF) receptors (FGFRs) in the Shionogi 115 (S115) mouse mammary tumor cell line and its genetic variant Clone 22. In S115 cells, androgen maintains a transformed morphology, rate of proliferation, and serum and anchorage independence. Similar effects were induced by treatment of the cells with FGF-2 or a heparin-binding growth factor (HBGF) fraction prepared from the medium conditioned by the cells. The effects of androgen and FGF-2 could be partly reversed with a specific anti-FGF-2 immunoglobulin G or by suramin, which inhibits binding of FGFs to their high affinity receptors. Testosterone and FGF-2 increased the expression of FGFR-1 messenger RNA (mRNA) and, to a lesser extent, FGFR-3 mRNA, but down-regulated FGFR-2 mRNA in S115 cells. No FGFR-4 mRNA was detected. FGF-2 also down-regulated the expression of syndecan-1, a heparan sulfate proteoglycan that binds FGF with low affinity. The binding of radiolabeled FGF-2 to FGFRs was lower in the cells cultured with testosterone or in the presence of the HBGFs from androgen-treated cells, presumably because of the autocrine production of FGF-like factors. In Clone 22 cells, FGFRs and syndecan-1 responded to androgen as in S115 cells, but they were less sensitive to FGF-2. Androgen or FGF-2 could not induce morphological transformation, although both stimulated proliferation. Androgen-increased proliferation was not, however, decreased by anti-FGF-2 immunoglobulin G in Clone 22 cells. These data suggest that of the HBGFs produced, FGF-2 is required in androgen induction of morphological change, whereas the effect on proliferation involves other factors as well (perhaps mostly FGF-8). The results show that androgen differentially regulates the expression of the high and low affinity FGF receptors, which could mediate androgen induction of the transformed phenotype in S115 cells by an autocrine mechanism. The differential responses of the Clone 22 variant cells to androgen and FGF-2 suggest that the pathways of steroid induction of different parameters of the transformed phenotype, such as transition to fibroblastic morphology and stimulation of proliferation, are divergent.

Animals↗
PubMed2004

[Establishment of assay system for fibroblast growth factor (FGF)-23 and pathophysiological roles of FGF-23 in the development of hypophosphatemic diseases].

Rickets/osteomalacia is characterized by impaired mineralization of bone matrix. X-linked hypophosphatemic rickets/osteomalacia(XLH), autosomal dominant hypophosphatemic rickets/osteomalacia(ADHR) and tumor-induced rickets/osteomalacia(TIO) share common clinical features including impaired proximal tubular phosphate reabsorption. Fibroblast growth factor(FGF)-23 was positionally cloned as a responsible gene for ADHR. We have also cloned FGF-23 as a causative factor for TIO. FGF-23 is a polypeptide with 251 amino acids. FGF-23 is proteolytically cleaved between Arg179 and Ser180 and only full-length FGF-23 showed biological activity to induce hypophosphatemia. Using two monoclonal antibodies that recognize N-terminal and C-terminal portion of the processing site of FGF-23, sandwich enzyme-linked immunosorbent assay for FGF-23 was established. This assay detects only full-length FGF-23. Circulatory level of FGF-23 in healthy controls distributed between 10 to 50 pg/ml suggesting that FGF-23 is a physiological humoral factor. FGF-23 levels in patients with TIO were elevated and rapidly decreased after removal of responsible tumors for TIO. These results confirm that FGF-23 is a causative factor of TIO and indicate that measurement of FGF-23 is useful for diagnosis and management of TIO. Moreover, FGF-23 levels were elevated in most patients with XLH suggesting FGF-23 is also involved in the pathogenesis of XLH. Further analysis is required to clarify more detailed actions of FGF-23 and its roles in the development of other diseases with disordered phosphate metabolism.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

Fibroblast growth factor receptor-1 is a critical component for endometrial remodeling: localization and expression of basic fibroblast growth factor and FGF-R1 in human endometrium during the menstrual cycle and decreased FGF-R1 expression in menorrhagia.

Angiogenic growth factors play a critical role in the cyclic growth and vascularization of normal endometrium. Herein, we report the expression and localization of both basic fibroblast growth factor (FGF-2) and its receptor (FGF-R1; flg) in human endometrium and demonstrate the markedly decreased FGF-R1 levels in menorrhagia. In situ hybridization using [35S]-labeled riboprobe demonstrated distinct autoradiographic signals for FGF-2 mRNA in glandular epithelial and stromal cells in endometrium throughout the menstrual cycle, with the strongest hybridization signal in stromal cells of the proliferative endometrium relative to that of the secretory endometrium. Moreover, RNAse protection assay revealed that the mRNA encoding FGF-2 and FGF-R1 was significantly higher in proliferative than in secretory endometrium (p < 0.05, p < 0.01). Immunohistochemistry using anti-flg antibody showed that the intensity of FGF-R1 staining was markedly diminished in the stromal cells of secretory endometrium, which corresponded with the reduced FGF-2 mRNA expression. In contrast, the endometrial glandular epithelial cells showed intense localization of FGF-R1 protein throughout the menstrual cycle, which paralleled FGF-2 mRNA expression. Colocalization of FGF-2 and FGF-R1 in stroma and stimulation of DNA synthesis and phospholipase C activation by FGF-2 in these cells demonstrates that FGF-2 acts in an autocrine manner in endometrial stroma. Western immunoblotting showed that FGF-R1 immunoprotein was markedly reduced or absent in women with menorrhagia throughout the cycle relative to that of normal cycling women, suggesting that FGF-R1 is critical for endometrial "maturation" and regeneration of the normal endometrium following menstruation.

Adult↗
PubMed1998

Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate.

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.

Animals↗
PubMed1997

Is the sensitivity of cells for FGF-1 and FGF-2 regulated by cell surface heparan sulfate proteoglycans?

We have examined the importance of cell surface heparan sulfate proteoglycans (HSPG) in fibroblast growth factor (FGF) signaling. 3T3 cells grown under conventional conditions were much more sensitive to FGF-2 compared to FGF-1. However, cells were equally sensitive to FGF-1 and FGF-2 using conditions which reduced the effect of endogenous HSPG. Addition of heparin, or treatment with chlorate, an inhibitor of proteoglycan sulfation, resulted in enhanced or reduced growth factor response, respectively, and eliminated the differences between FGF-1 and FGF-2. HSPGs isolated from trypsin digests of 3T3 cells had a much higher affinity for FGF-2 compared to that for FGF-1 when analyzed by affinity chromatography. Glycosaminoglycan chains or core protein fragments derived from the HSPG failed to show the same high apparent affinity for FGF-2, suggesting that an intact proteoglycan structure was important for the high FGF-2 affinity. Addition of HSPG ectodomains, isolated from cultured 3T3 cells or produced as recombinant molecules, to chlorate-treated cultures of 3T3 cells inhibited the mitogenic activity of FGF-2 and eliminated the effect of heparin as a potentiator of either growth factor. These results support the idea that the cell-associated HSPG is an integral component of the FGF signaling system and in 3T3 cells contributes to the increased sensitivity of these cells to FGF-2 compared to FGF-1. Since isolated ectodomains of HSPG inhibited rather than stimulated the mitogenic response of the FGFs, the proper anchoring of the HSPG in the cell membrane appears to be important for a stimulatory effect.

3T3 Cells↗
PubMed2000

Fibroblast growth factors (FGF-1, FGF-2) promote migration and neurite growth of mouse cochlear ganglion cells in vitro: immunohistochemistry and antibody perturbation.

To study the effect of FGF in the early development of the sensory neurons of the auditory system, we established a culture preparation of ganglionic neuroblasts engaged in migration and process outgrowth. The presumed anlage of the cochlear ganglion was dissected from E11 otocysts, just as the neuronal precursors were migrating. The cultures were divided into 4 groups and supplemented for 7-9 days with either hrFGF-1 or hrFGF-2 or both or with defined medium only (control group). Measurements of the increase in explant growth, neuroblast migration, and neurite outgrowth were made by time-lapse imaging techniques in living cultures. Either FGF-1 or FGF-2 alone stimulated early migration and outgrowth of the ganglion cells by 5-10x. The effect of combining FGF-1 and FGF-2 was greater than either alone, but less than additive, consistent with a shared receptor. BrdU labeling confirmed that the effect was on migration, not on proliferation. Adding a neutralizing antibody for FGF-2 to the cultures inhibited migration and neurite outgrowth, suggesting an endogenous FGF-2 activity in these functions. Immunocytochemical observations in vitro and in situ with antibodies to FGF-1, FGF-2, or FGF receptor (R1) demonstrated immunopositive staining of the migrating ganglionic neuroblasts, their processes, and growth cones at corresponding stages (E13). Also non-neuronal cells, hair cells, and Schwann cells (in situ) expressed FGF-1 and FGF-2. Evidently both FGF-1 and FGF-2 play important roles in the migration and initial differentiation of cochlear ganglion neurons in the mouse.

Animals↗
PubMed1995

Expression of FGF-2 and FGF receptor type 1 in the adult rat brainstem: effect of colchicine.

In the adult rat brainstem, neuronal subpopulations of several motor and sensory nuclei display basic fibroblast growth factor (bFGF or FGF-2) immunoreactivity (IR; Grothe et al. J. Comp. Neurol. 305:328-336). In the present study we demonstrate that FGF-2-IR correlates with staining for the high-affinity FGF-receptor 1. Intracerebroventricular injection of colchicine leads to the disappearance or substantial reduction of FGF-2-IR in the hypoglossal, facial, trigeminal motor, trochlear, and mesencephalic trigeminal nuclei. In contrast, FGF-2-IR appears in many perikarya of the red nucleus and the medial nucleus of the trapezoid body, whereas in control rats both nuclei showed immunostained fibers and almost no immunoreactive cell bodies. This dramatic change of FGF-2-IR could be explained by the ability of colchicine to block fast axonal transport. Cranial nuclei may internalize FGF-2 at the periphery via high-affinity receptors and retrogradely transport the molecule to their perikarya. The red nucleus and the medial nucleus of the trapezoid body may synthesize FGF-2 and provide the growth factor to afferent or efferent neurons. The presence of FGF-2 mRNA in brainstem extract and the absence of the FGF-2 transcript in extracts of the hypoglossal nucleus corroborate this suggestion. The effect of colchicine on FGF-2-IR in the brainstem nuclei suggests that FGF-2 could be specifically retrogradely transported in other cranial nuclei, in addition to the hypoglossal system. Together with the ability of FGF-2 to stimulate neuronal survival, this result strongly supports the hypothesis that FGF-2 is acting as a neurotrophic factor on specific central neuron populations.

Animals↗
PubMed2003

FGF-2 but not FGF-1 binds fibrin and supports prolonged endothelial cell growth.

Endothelial cell viability and growth are dependent on both polypeptide growth factors, and integrin-mediated matrix interactions. We have now examined the ability of fibrin-binding and non-binding growth factors to support long-term endothelial cell growth in the presence or absence of the soluble form. Endothelial cells were cultured on a fibrin surface, with or without FGF-1 or FGF-2, and proliferation was determined by (3)H-thymidine incorporation. Cells cultured on fibrin with no growth factor showed minimal proliferation up to 96 h. In contrast, when FGF-2 was incorporated into fibrin, proliferation was increased 6.5 +/- 0.6-fold, equal to growth on a fibrin surface with FGF-2 continually present in the medium. Thymidine incorporation was similar when cells were cultured on a fibrin surface that had been incubated with FGF-2 and then the growth factor removed (8.6 +/- 0.5-fold). In contrast to results with FGF-2, a surface of fibrin exposed to FGF-1 supported minimal growth, whereas growth was comparable to either FGF-1 or FGF-2 present in the medium. Comparable results were observed when proliferation was quantitated by cell counting at times up to 48 h. Binding studies demonstrated no high-affinity interaction of FGF-1 with fibrinogen or fibrin. We conclude that FGF-2 bound to fibrin supports prolonged endothelial cell growth as well as soluble FGF-2, whereas FGF-1 does not bind to fibrin and can support endothelial cell growth only if continually present in soluble form. Fibrin may serve as a matrix reservoir for FGF-2 to support cell growth at sites of injury or thrombosis.

Cell Division↗
PubMed1997

Expression of FGF-1 and FGF-2 mRNA during lens morphogenesis, differentiation and growth.

PURPOSE: There is now considerable evidence that FGF is involved in lens differentiation and growth throughout life. The aim of this study was to determine potential sites of FGF production in and near the lens during morphogenesis, differentiation and growth. METHODS: The distribution of FGF-1 and FGF-2 mRNAs was analysed in embryonic, weanling and adult rat eyes by in situ hybridization. RESULTS: During lens morphogenesis, there was distinct expression of FGF-1, but not FGF-2, in the lens placode and retinal disc cells. Subsequently, both forms of FGF showed similar expression patterns. During lens differentiation, distinct expression of FGFs was associated with elongating primary fiber cells. From embryonic day 20 onwards, lenses showed strongest expression of FGF mRNAs in the transitional zone, where epithelial cells differentiate into fibers, with weaker expression in the anterior epithelium. Messenger RNAs for both FGFs were also localised in ocular tissues near the lens and bordering the ocular media, particularly the cornea, ciliary body, iris and neural retina. CONCLUSIONS: These findings are consistent with the known distribution of FGF protein in the eye and implicate various ocular tissues as potential sources of FGF that may influence lens cells. Furthermore, the fact that lens cells have the potential for synthesizing FGF, together with evidence from previous studies that lens cells express FGF receptors and respond to lens-derived FGF, raises the possibility that some aspects of lens cell behaviour in situ may be influenced by autocrine mechanism(s) of FGF stimulation.

Animals↗
PubMed2001

Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition.

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4-HS complexes, FR1c fails to bind FGF-4-HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.

Alkaline Phosphatase↗
PubMed2003

Functions of fibroblast growth factor (FGF)-2 and FGF-5 in astroglial differentiation and blood-brain barrier permeability: evidence from mouse mutants.

Multiple evidence suggests that fibroblast growth factors (FGFs), most prominently FGF-2, affect astroglial proliferation, maturation, and transition to a reactive phenotype in vitro, and after exogenous administration, in vivo. Whether this reflects a physiological role of endogenous FGF is unknown. Using FGF-2 and FGF-5 single- and double mutant mice we show now a region-specific reduction of glial fibrillary acidic protein (GFAP), but not of S100 in gray matter astrocytes. FGF-2 is apparently the major regulator of GFAP, because in mice deficient for FGF-2, GFAP is distinctly reduced in cortex and striatum, whereas in FGF-5-/- animals only a reduction in the midbrain tegmentum can be observed. In FGF-2-/-/FGF-5-/- double mutant animals, GFAP-immunoreactivity is reduced in all three brain regions. Cortical astrocytes cultured from FGF-2-/-/FGF-5-/- double mutant mice revealed reduced levels of GFAP, but not S100 as compared with wild-type littermates. This phenotype could be rescued by exogenous FGF-2 but not FGF-5 (10 ng/ml). Electron microscopy revealed reduced levels of intermediate filaments in perivascular astroglial endfeet. This defect was accompanied by enhanced permeability of the blood-brain barrier (BBB), as detected by albumin extravasation. Levels of the tight junction proteins Occludin and ZO-1 were reduced in blood vessels of FGF-2-/-/FGF-5-/- double mutant mice as compared with wild-type littermates. Our data support the notion that endogenous FGF-2 and FGF-5 regulate GFAP expression in a region-specific manner. The observed defect in astroglial differentiation is accompanied by a defect in BBB function arguing for an indirect or direct role of FGFs in the regulation of BBB permeability in vivo.

Animals↗
PubMed1999

Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells.

Basic fibroblast growth factor (FGF-2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF-2 and FGF receptors (FGFR-1 to -4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF-2 and FGFR in vitro and in vivo. FGF-2 expression did correlate with a short doubling time as well as with potent anchorage-independent growth of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF-2 into the extracellular space. Expression levels of FGFR-1 and -2 in NSCLC cell lines correlated with FGF-2 production. FGFR were located at the plasma membranes in some low FGF-2-producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF-2 (rFGF-2). In NSCLC cell lines with an enhanced FGF-2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF-2 and growth inhibition by FGF-2-neutralising antibodies. In contrast, several agents antagonised FGF-2 intracellularly impaired growth of almost all NSCLC cell lines. Our data suggest a role of FGF-2 and FGFR in the growth stimulation of NSCLC cells possibly via an intracrine mechanism.

3T3 Cells↗
PubMed2000

An overexpression of fibroblast growth factor (FGF) and FGF receptor 4 in a severe clinical phenotype of facioscapulohumeral muscular dystrophy.

We evaluated the expression of a select panel of growth factors and their receptors, including fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor (PDGF), FGF receptor 1 (FGF-R1), FGF receptor 3 (FGF-R3), FGF receptor 4 (FGF-R4), PDGF receptor alpha (PDGF-Ralpha), PDGF receptor beta (PDGF-Rbeta), and heparan sulfate proteoglycan (HSPG), in muscle biopsy specimens from nine facioscapulohumeral muscular dystrophy (FSHD) patients using immunohistochemistry. Two cases of Duchenne-type muscular dystrophy (DMD), two of Becker-type muscular dystrophy (BMD), and one of limb-girdle-type muscular dystrophy (LGMD) were also investigated. Widespread immunostaining for FGF-1 and FGF-2 on the sarcolemma and overexpression of FGF-R4 in endomysial and perimysial connective tissue were seen in one patient with a severe clinical phenotype of FSHD who had respiratory failure. Standard histochemistry in this patient revealed marked interstitial fibrosis and lobulated fibers. The overexpression of FGF and FGF-R4 in this severe FSHD case may be associated with the muscle fibrosis and disease severity.

Adolescent↗
PubMed2000

Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits.

We have examined the relationship between Na,K-ATPase and FGF-2 secretion in transfected primate cells. FGF-2 lacks a classic hydrophobic export signal, and the mechanisms mediating its secretion are unknown. To monitor secretion, a FLAG epitope tag was inserted into the carboxyl terminus of the 18 kDa form of human FGF-2, and the construct was transfected into either human HEK 293 or monkey CV-1 cells. Exported FGF-2 was detected in the culture medium using the FLAG-specific monoclonal antibody M2. FGF-2 secretion from HEK 293 or CV-1 cells was linear over time and sensitive to inhibition by the cardiac glycoside ouabain, a specific inhibitor of the Na,K-ATPase. In contrast, the secretion of FGF-8 (an FGF family member that contains a hydrophobic secretory signal) was not inhibited by treatment of HEK 293 or CV-1 cells with ouabain. FGF-2 secretion was also assayed in CV-1 cells expressing the naturally ouabain-resistant rodent Na,K-ATPase alpha1 subunit. In cells expressing the rodent alpha1 subunit, FGF-2 secretion was unaffected by high levels of ouabain, indicating that the rodent alpha1 subunit was capable of rescuing ouabain-inhibitable FGF-2 export. Expression of ouabain-resistant mutants of the rodent alpha2 and alpha3 subunits, or the naturally ouabain-resistant rodent alpha4 subunit, also supported FGF-2 secretion in ouabain-treated cells. Taken together, our studies are consistent with the idea that the Na,K-ATPase plays a prominent role in regulating FGF-2 secretion, although none of the alpha subunit isoforms exhibited specificity with regard to FGF-2 export.

Animals↗
PubMed2002

Ribozyme-targeting of a secreted FGF-binding protein (FGF-BP) inhibits proliferation of prostate cancer cells in vitro and in vivo.

Prostate cancer is one of the most common malignant tumors with increasing incidence rates in the aging male. Since locally advanced or metastatic prostate tumors are essentially incurable, identification of new target molecules and treatment strategies is of critical importance. Fibroblast growth factor-2 (FGF-2) acts as potent mitogen which is upregulated in prostate cancers modulating cancer cell proliferation and development of an invasive phenotype. Normally it is tightly bound to the extracellular matrix that quenches its biological activity. The FGF-binding proteins (FGF-BP, HBp17) is a secreted protein which is able to mobilize and activate FGF-2 from the extracellular matrix. Here we show that FGF-BP is highly expressed in prostate tumor cells. To study the functional role of FGF-BP, we use a ribozyme-targeting approach to selectively deplete FGF-BP in prostate cancer cells achieving a more than 50% reduction of FGF-BP mRNA and protein levels in two mass-transfected cell lines. FGF-BP depletion reduces proliferation of the cells in vitro without changes in cell cycle distribution or apoptosis. Using cDNA microarrays, Northern blotting and RT-PCR, we show a complex pattern of changes in the gene expression profiles upon FGF-BP depletion. Most strikingly, ribozyme-mediated reduction of FGF-BP levels completely abolishes the ability of the highly metastatic PC-3 prostate carcinoma cells to grow tumors in an athymic nude mouse in vivo model which is far beyond the effects of FGF-BP ribozyme targeting observed previously in cells from other tumors in the same model. Taken together, our study identifies FGF-BP as a potential rate-limiting factor for prostate cancer growth and, due to its restricted expression pattern in adults, a potentially attractive target for prostate cancer therapy.

Apoptosis↗