[Involvement of FGF-FGF receptor paracrine system and homeobox gene products in the formation of axial mesodermal structures in Xenopus embryos].
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Animals↗
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[FGF receptor and FGF signaling pathways].
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Animals↗
Regulation of FGF receptors in the oligodendrocyte lineage.
Fibroblast growth factors (FGFs) affect a broad spectrum of developmentally regulated cellular responses involved in the control of growth and differentiation. To identify specific FGF receptor forms involved in these responses, we have characterized FGF receptor transcript expression, and its modulation by FGF-2, as enriched populations of oligodendrocyte progenitors differentiate into mature oligodendrocytes. The data demonstrate that the levels of mRNA expression for FGF high-affinity receptors-1, -2, and -3 are differentially regulated during lineage progression: FGF receptor-1 expression increases with lineage progression, FGF receptor-2 is predominantly expressed by terminally differentiated oligodendrocytes, and FGF receptor-3 reaches a peak level of expression in late progenitors and then declines upon further differentiation; FGF receptor-4 expression was not detected in oligodendrocytes. Distinct patterns of alternatively spliced variants of FGF receptor-1 and -2 transcripts are expressed: the predominant FGF receptor-1 transcripts contain three Ig-like domains (FGF receptor-1 alpha), whereas the FGF receptor-2 transcripts contain two Ig-like domains (FGF receptor-2 beta 2) and this form is up-regulated as oligodendrocytes differentiate. In addition, the expression of these receptors is differentially regulated by the ligand, FGF-2: FGF receptor-1 mRNA expression is up-regulated in early progenitors, and FGF receptor-2 mRNA expression is down-regulated in mature oligodendrocytes. Finally, astrocytes express FGF receptor-1, -2, and -3 transcripts, but at different levels and with different exon utilization (FGF receptor-1 beta, FGF receptor-2 beta 1/beta 2) compared to oligodendrocytes. To our knowledge this is the first report that demonstrates that the mRNA expression of these three FGF receptor types is differentially regulated in primary cells as they differentiate along a lineage from progenitors to terminally differentiated cells. We propose that this pattern of expression provides a molecular basis for the developmentally varying response of cells to a common ligand. For example, according to this hypothesis, in response to FGF-2, FGF receptor-1 transduces signals that stimulate the prolonged proliferation and migration of early progenitors, FGF receptor-3 promotes the proliferation and arrest of differentiation of late progenitors, and FGF receptor-2 transduces signals for terminal differentiation, but not proliferation, in mature oligodendrocytes.
Alternative Splicing↗
Molecular cloning and characterization of human FGF-20 on chromosome 8p21.3-p22.
The fibroblast growth factors (FGFs) play important roles in morphogenesis, angiogenesis, tissue remodeling, and carcinogenesis. Human FGF-20 has been cloned and characterized in this study. FGF-20 encodes a 211-amino-acid polypeptide with the FGF-core domain. A strong hydrophobic region was found in the FGF-core domain of FGF-20; however, no typical N-terminal signal sequence was found in FGF-20, just as in FGF-9 and FGF-16. Total amino acid identities are as follows: FGF-20 vs FGF-9, 71.6%; FGF-20 vs FGF-16, 66.2%; FGF-9 vs FGF-16, 72.4%. Phylogenic analysis indicated that FGF-20, FGF-9, and FGF-16 constitute a subfamily among the FGF family. FGF-20 mRNA of 2.4 kb in size was detected in colon cancer cell line SW480 by Northern blot analysis. Lower levels of FGF-20 mRNA were detected in human fetal tissues and primary cancers by cDNA-PCR. The nucleotide sequence of FGF-20 cDNA is split into three parts in the human genome sequence of the chromosome 8p21.3-p22 region (Accession No. AB020858). These results indicate that the FGF-20 gene, located on human chromosome 8p21.3-p22, consists of three exons. Compared with the nucleotide sequence of FGF-20 cDNA determined in this study, one nucleotide deletion and one nucleotide substitution in the putative coding region were identified in human genome sequence AB020858.
Alternative Splicing↗
Regulation of C-terminal and intact FGF-23 by dietary phosphate in men and women.
UNLABELLED: FGF-23 is a novel regulator of phosphate metabolism. We studied the regulation of FGF-23 by dietary phosphate in 66 men and women using two assays. Dietary phosphate restriction decreased FGF-23 and loading increased FGF-23 significantly. An assay that measured intact FGF-23 showed the effects of dietary phosphate much more clearly than an assay that also measures presumed biologically inactive fragments. Dietary phosphate is a key regulator of circulating FGF-23; choice of assay is critical when studying FGF-23 physiology. INTRODUCTION: Fibroblast growth factor 23 (FGF-23) is a novel phosphaturic factor discovered through genetic studies of patients with renal phosphate wasting disorders. Ablation of the FGF-23 gene in mice reduces renal phosphate excretion and increases serum phosphate, suggesting that FGF-23 is critical for normal phosphate homeostasis. We examined the role of dietary phosphate in the regulation of FGF-23 in humans. MATERIALS AND METHODS: Sixty-six healthy males and females were randomized to either phosphate-depleted or -loaded diets for 5 days, after a 4-day run-in diet. FGF-23 was measured using an "intact" assay that only detects intact FGF-23 peptide and with a "C-terminal" assay that measures both intact FGF-23 peptide and presumed biologically inactive carboxyl terminal fragments. The main outcome was the within group change in FGF-23 with either phosphate depletion or loading. RESULTS: Using the intact FGF-23 assay, mean FGF-23 area under the curve (AUC) decreased by 9 +/- 16% with phosphate depletion (p = 0.0041) and increased by 35 +/- 29% with loading (p < 0.0001). Using the C-terminal FGF-23 assay, mean FGF-23 AUC decreased by 8 +/- 12% with phosphate depletion (p = 0.0003) and increased by 13 +/- 20% with loading (p = 0.0016). Increases in FGF-23 with phosphate loading were greater with the intact assay than with the C-terminal assay (p = 0.0003). Using the intact assay only, FGF-23 was significantly associated with serum phosphate (r = 0.39, p < 0.01), 24-h urinary phosphate (r = 0.47, p < 0.01), fractional excretion of phosphate (r = 0.29, p < 0.01), and 1,25-dihydroxyvitamin D (r = -0.30, p < 0.01). The association between the assays was weak (r = 0.26, p < 0.01). CONCLUSIONS: Dietary phosphate is a key regulator of circulating FGF-23 levels in humans. Additionally, choice of assay is critical when performing physiologic investigations of FGF-23.
Adolescent↗
Secretion of basic fibroblast growth factor (FGF-2) by WEHI-3B myelomonocytic leukemia cells.
In order to investigate the role of Fibroblast Growth Factors in hematopoietic cells, we studied the expression of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5 and FGF-6 mRNAs both in murine myelomonocytic leukemia WEHI-3B and in a murine stromal cell line SR-4987. Secretion of FGF-2 in the cell culture supernatant was also studied. Expression of mRNA encoding for the above-mentioned FGFs was analyzed by RT-PCR. The production of FGF-2 in the conditioned media of WEHI-3B and SR-4987 cell cultures was evaluated by techniques of affinity chromatography, chromatofocusing and immunoblotting. The biological activity of FGF-2 was checked on SR-4987 cells by a agar clonogenic assay. In both cell lines mRNA was found encoding for FGF-1, FGF-2 and FGF-6 and WEHI-3B cells express also mRNA for FGF-3 (int-2) and FGF-4 (K-FGF/hst). Furthermore, supernatant from WEHI-3B cells was found to stimulate dramatically the agar clonogenicity of SR-4987 cells which have a very poor basal capacity for growth in agar. The clonogenic activity of WEHI-3B conditioned medium is due to FGF-2 secreted into cell culture supernatant whereas SR-4987 cells, although express FGF-2 mRNA, do not seem able to secrete this factor. The expression in myeloid leukemia cells of oncogene-related factors such as FGF-3, FGF-4 and FGF-6 together with the secretion of FGF-2 able to support a positive regulation of bone marrow stromal cells function suggest that FGFs may have an important role in sustaining the leukemogenic process and related disorders.
Animals↗
Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures.
Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), FGF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.
Animals↗
Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain.
We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.
Amino Acid Sequence↗
Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells.
Basic fibroblast growth factor (FGF-2) is a secretory protein that lacks a signal peptide. Consistently, FGF-2 has been shown to be secreted by an ER-Golgi-independent mechanism; however, the machinery mediating this process remains to be established at the molecular level. Here we introduce a novel experimental system based on flow cytometry that allows the quantitative assessment of non-classical FGF-2 secretion in living cells. Stable cell lines have been created by retroviral transduction that express various kinds of FGF-2-GFP fusion proteins in a doxicyclin-dependent manner. Following induction of protein expression, biosynthetic FGF-2-GFP is shown to translocate to the outer surface of the plasma membrane as determined by both fluorescence activated cell sorting (FACS) and confocal microscopy. Both N- and C-terminal GFP tagging of FGF-2 is compatible with FGF-2 export, which is shown to occur in a controlled fashion rather than through unspecific release. The experimental system described has strong implications for the identification of both FGF-2 secretion inhibitors and molecular components involved in FGF-2 secretion. In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is rate-limiting.
Animals↗
Inactivation of the FGF-4 gene in embryonic stem cells alters the growth and/or the survival of their early differentiated progeny.
Previous studies have shown that early mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, we prepared genetically engineered embryonic stem (ES) cells, which are unable to produce FGF-4. Specifically, we describe the isolation and characterization of ES cells with both FGF-4 alleles inactivated. The FGF-4-/- ES cells do not require FGF-4 to proliferate in vitro, and addition of FGF-4 to the medium has little or no effect on their growth. Thus, FGF-4 does not appear to act as an autocrine growth factor for cultured ES cells. We also demonstrate that FGF-4-/- ES cells, like their unmodified counterparts, are capable of forming highly complex tumors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, we demonstrate that the FGF-4-/- ES cells can differentiate in vitro after exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importantly, addition of FGF-4 to the culture medium dramatically increases the number of differentiated cells derived from the FGF-4-/- ES cells, in particular cells with many of the properties of parietal extraembryonic endoderm. Finally, we demonstrate that there are differences in the RNA profiles expressed by the differentiated progeny formed in vitro from FGF-4-/- ES cells and FGF-4+/+ ES cells when they are cultured with FGF-4. Taken together, the studies described in this report indicate that certain lineages formed in vitro are affected by the inactivation of the FGF-4 gene, in particular specific cells that form during the initial stage of ES cell differentiation. Thus, ES cells with both FGF-4 alleles inactivated should shed light on the important roles of FGF-4 during the early stages of mammalian development and help determine why FGF-4-/- embryos die shortly after implantation.
Animals↗
Decline in basic fibroblast growth factor (FGF-2) mRNA expression in female rat hypothalamus at puberty.
There is a growing acceptance of the importance of hypothalamic growth factors in the control of sexual maturation. Basic fibroblast growth factor (bFGF, FGF-2), a potent mitogen and neurotropic factor for brain cells in vitro, including hypothalamic cells, is widely expressed in the post-natal CNS but its physiological functions there are largely unknown. Previously, studies of FGF-2 mRNA regulation in vivo have been hampered by the low levels of FGF-2 mRNA present in post-natal tissues. We have applied a sensitive semi-quantitative procedure based on reverse transcription followed by polymerase chain reaction amplification (RT-PCR) to detect and estimate relative amounts of mRNAs encoding FGF-2 and its receptor in the hypothalamic-hypophyseal axis in individual female rats undergoing sexual maturation. FGF receptor and FGF-2 mRNAs were detectable in all brain regions examined. Injections of the glutamate agonist N-methyl-D-aspartic acid (NMDA) or pregnant mare's serum gonadotropin (PMSG) were used to advance the onset of puberty in immature female rats, and the levels of FGF-2 and FGF receptor mRNA in MBH and cortex were examined. Daily injections of NMDA (20 mg/kg) from day 24-28 resulted in advancement of first ovulation and vaginal opening (VO) in 5 of 9 treated rats. None (0/4) of the saline treated controls achieved first ovulation during the course of the experiment. Expression of FGF-2 mRNA in the medial-basal hypothalamus of the NMDA-treated VO animals, but not nonVO animals, was significantly (P<0.05) reduced by 50% vs saline-treated nonVO controls. There was no effect of NMDA on FGF-2 expression in cerebral cortex of VO Vs nonVO animals. FGF receptor mRNA levels were unaffected by NMDA treatment. To assess the possibility that the decline in hypothalamic FGF-2 mRNA levels was related to puberty and not just to an effect of NMDA, pregnant mare's serum gonadotropin was used to induce first ovulation and vaginal opening. Injection of PMSG to immature female rats on day 26 resulted in precocious first ovulation on day 29. This was accompanied by a significant 40% reduction in the steady-state level of FGF-2 mRNA in the medial basal hypothalamus compared to saline treated controls. As with NMDA treatment, PMSG did not affect FGF-2 mRNA abundance in the cortex, nor the FGF receptor mRNA in MBH or cortex. Immunohistochemical detection of FGF-2 protein in the arcuate nucleus revealed that FGF-2 immunoreactivity was also significantly modified in peri-ovulatory NMDA-treated animals. FGF-2 immunoreactivity in NMDA treated rats was significantly elevated at day 29 (the day of ovulation), but significantly inhibited by day 33. These findings suggest that alterations in the level of FGF-2 mRNA in the hypothalamus may be associated with first ovulation and the onset of sexual maturation in the female rat.
Animals↗
Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines.
We have previously reported the tissue specific distribution of four different FGF-1 transcripts containing alternative 5' untranslated exons spliced to the first protein coding exon. The predominant transcript in brain is FGF-1.B and in kidney FGF-1.A. Others have shown, by in situ hybridization and immunohistochemical analysis, that expression of FGF-1 in the brain is exclusively in neural cells but not in glial cells. Here we have examined the distribution of FGF-1.B and FGF-1.A transcripts in glioblastoma and retinal tissues and in kidney carcinoma cell lines. Our results show that FGF-1.B is the predominant transcript in neural derived tissues including both the diabetic retina and normal retina tissues. Surprisingly, FGF-1.B transcript is highly expressed in glioblastoma tissues. In contrast, a normal brain glial cell line, CHII, expresses very low levels of FGF-1 mRNA. These results strongly implicate the role of FGF-1 in the etiology of glioblastoma. We also examined several kidney carcinoma derived cell lines for the expression of FGF-1 mRNA. Most of these kidney cell lines do not express any FGF-1 transcripts. An interpretation by deduction is that kidney adenocarcinomas are derived from cortex but medulla has been reported as the site of FGF-1 synthesis. Of the kidney derived cell lines which are positive for FGF-1 message, only one expressed FGF-1.A transcript. The data may suggest that the establishment of kidney cell lines results in a switch of promoter usage from the 1.A seen in kidney tissue. Similarly, culturing of glioma cell lines may result in a switch from FGF-1.B seen in glioma tissues to FGF-1.D seen in most glioma cell lines. Continued studies of the FGF-1 transcripts, their functional promoters and their tissues distribution will provide insight into the potential role of FGF-1 in cell growth, tissue differentiation and malignant transformation.
Alternative Splicing↗
FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain.
We have isolated cDNA encoding a novel FGF (212 amino acids) from rat brain. Because this is the 20th documented member of the FGF family, we tentatively term it FGF-20. Among FGF family members, FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62% amino acid identity, respectively). Human FGF-20 gene was found in the human genomic sequence mapped to the 8p21.3-p22 region. Human FGF-20 is highly identical to rat FGF-20 (95% amino acid identity). FGF-20 mRNA was preferentially expressed in rat brain among the adult major tissues examined. The localization of FGF-20 mRNA in rat brain was also examined by in situ hybridization. FGF-20 mRNA was preferentially expressed in the substantia nigra pars compacta. To examine the biological activity of FGF-20, recombinant rat FGF-20 was produced by insect cells infected with recombinant baculovirus containing rat FGF-20 cDNA. Recombinant rat FGF-20 enhanced the survival of midbrain dopaminergic neurons. The present results indicate that FGF-20 is a novel neurotrophic factor preferentially expressed in the substantia nigra pars compacta of rat brain.
Amino Acid Sequence↗
Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF-18.
We isolated the cDNA encoding a novel member (207 amino acids) of the fibroblast growth factor (FGF) family from rat embryos. Because this protein is the 18th documented member of the FGF family, we tentatively termed it FGF-18. We have also determined mouse and human FGF-18 with high amino acid identity (99.5 and 99.0%) to rat FGF-18, respectively. Among FGF family members, FGF-18 is most similar (52.7% amino acid identity) to FGF-8 and FGF-17. FGF-18 has a typical signal sequence at its amino terminus. Recombinant rat FGF-18, which was efficiently secreted by High Five insect cells infected with recombinant baculovirus containing the cDNA, induced neurite outgrowth in PC12 cells. The expression of FGF-18 mRNA was examined in adult rat tissues and embryos by Northern blotting analysis and in situ hybridization. FGF-18 mRNA of approximately 2. 7 kilobases was preferentially detected in the lung among adult rat tissues examined. In rat embryos, FGF-18 mRNA was detected in several discrete regions at embryonic days 14.5 and 19.5 but not at E10.5. The temporal and spatial patterns of FGF-18 mRNA expression in embryos are quite different from those of FGF-8 and FGF-17 mRNAs reported. The present results indicate that FGF-18 is a unique secreted signaling molecule in the adult lung and developing tissues.
Amino Acid Sequence↗
Immunolocalization of fibroblast growth factor-1 (FGF-1), its receptor (FGFR-1), and fibroblast-specific protein-1 (FSP-1) in inflammatory renal disease.
BACKGROUND: The fibroblast growth factor (FGF) family has functions in development, cell proliferation, migration, and differentiation. While FGF-2 induces fibrosis, the role of FGF-1 in inflammation and fibrosis is less defined. We examined the expression of FGF-1 and FGF receptor (FGFR-1) to determine if renal diseases with varying etiologies of inflammation, including lupus nephritis (LN), acute interstitial nephritis (AIN) and acute rejection superimposed on chronic allograft nephropathy (CAN), showed varying patterns of expression. We also examined the expression of fibroblast-specific protein-1 (FSP-1), which has been linked to epithelial-mesenchymal transition (EMT) and fibrosis, to determine whether it was linked to potential profibrotic and inflammatory FGF-1 mechanisms. METHODS: Proliferative LN (PLN) (N= 12), nonproliferative lupus nephritis (NPLN) (N= 5), AIN (N= 6), CAN (N= 4), and normal kidneys (N= 3) were studied. FGF, FGFR-1, and FSP-1 were localized by immunohistochemistry, and intensity scored on a 0 to 3+ scale. Double staining with CD68 and separate immunohistochemical staining for CD4 and CD8 with serial sections analysis were done to identify if T lymphocytes or macrophages showed staining for FGF-1 and FGFR-1 or FSP-1. RESULTS: In normal kidneys, FGF-1 was expressed in mesangial cells (0.67 +/- 0.58), glomerular endothelial (0.67 +/- 0.58), visceral, and parietal epithelial cells (1.67 +/- 0.58). FGFR-1 showed a similar pattern of staining but also was expressed in tubular epithelium, and arterial endothelium and smooth muscle. Expression of FGF-1 was increased over normal in glomerular parenchymal cells only in CAN in podocytes (2.30 +/- 0.58 vs. 3.00 +/- 0.00) (P < 0.05) and parietal epithelial cells (1.67 +/- 0.58 vs. 2.25 +/- 0.50) (P < 0.05). Infiltrating glomerular and interstitial inflammatory cells in diseased glomeruli also expressed FGF-1 and FGFR-1. Tubular cells expressed slightly increased FGFR-1 in renal diseases vs. normal, whereas tubules remained negative for FGF-1 in diseased kidneys. FSP-1 expression was prominent in the interstitium in all kidneys with interstitial inflammation, and most prominent in CAN. Interstitial FSP-1+ cells were consistent with a myofibroblast-type morphology, and did not stain with CD-68. FSP-1 expression was closely associated with inflammatory cells expressing FGF-1 and FGFR-1. FSP-1 also showed positivity within crescents and occasional podocytes in PLN. CONCLUSION: The expression of FGF-1 and FGFR-1 in infiltrating lymphocytes and macrophages, and of FGFR-1 in tubules, is supportive, but does not prove causality, of the possibility that FGF-1 might have both autocrine and paracrine functions in renal inflammation. However, the initial stimulus for renal inflammation, whether immune complex, hypersensitivity or rejection, did not alter expression patterns of FGF-1 or its receptor. The colocalization of inflammatory infiltrates with interstitial fibrosis supports the possibility of a contribution of FGF-1 for chemotaxis and associated fibrosis, further supported by interstitial FSP-1 expression closely associated with these inflammatory cells expressing FGF-1 and FGFR-1.
Acute Disease↗
Exogenous fibroblast growth factors maintain viability, promote proliferation, and suppress GADD45alpha and GAS6 transcript content of prostate cancer cells genetically modified to lack endogenous FGF-2.
Understanding processes regulating prostate cancer cell survival is critical to management of advanced disease. We used prostate cancer cell transfectants genetically modified to be deficient in either endogenous fibroblast growth factor (FGF-1) or endogenous FGF-2 to examine FGF maintenance of transfectant survival and proliferation and FGF-2-regulated expression of transfectant growth arrest DNA damage (GADD) and growth arrest sequences (GAS) family genes (known modulators of cell cycle progression and survival) and the AS3 gene (an androgen-modulated effector of prostate cell proliferation). When propagated in the absence of exogenous FGFs, FGF-2-deficient transfectants undergo exponential death, whereas FGF-1-deficient transfectants proliferate. Exogenous FGF-1, FGF-2, FGF-7, or FGF-8 promote survival and proliferation of FGF-2-deficient transfectants and enhance FGF-1-deficient transfectant proliferation. Transfectants express FGF receptor FGFR1, FGFR2(IIIb), FGFR2(IIIc), and FGFR3 transcripts, findings consistent with the effects of exogenous FGFs. FGF-2-deficient transfectants express high levels of AS3, GADD45alpha, GADD45gamma, GAS8, and GAS11 transcripts and moderate levels of GADD153, GAS2, GAS3, and GAS6 transcripts and lack demonstrable GAS1 or GAS5 transcripts. FGF withdrawal-mediated death of FGF-2-deficient transfectants did not significantly affect cell AS3, GADD153, GADD45gamma, GAS2, GAS3, GAS7, GAS8, or GAS11 transcript content, whereas GADD45alpha and GAS6 transcript content was elevated. These studies establish that endogenous FGF-2 dominantly regulates prostate cancer cell survival and proliferation and that exogenous FGFs may assume this function in the absence of endogenous FGF-2. Additionally, we provide the first evidence that FGFs regulate prostate GADD45alpha and GAS6 transcript content. The latter observations suggest that GADD45alpha and GAS6 proteins may be effectors of processes that regulate prostate cancer cell survival. Additional studies are required to examine this possibility in detail.
Animals↗
Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells.
The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.
3T3 Cells↗
Disregulation of ocular morphogenesis by lens-specific expression of FGF-3/int-2 in transgenic mice.
FGF-3, originally named int-2, was discovered as an oncogene frequently activated in mammary carcinomas resulting from the chromosomal integration of the mouse mammary tumor virus (MMTV). Int-2 was later designated FGF-3 based on sequence homology with other members of the fibroblast growth factor (FGF) family. FGF-1 is the prototypical member of the FGF family, and is the only family member which activates all known FGF receptor isoforms. Transgenic mice expressing in the lens a form of FGF-1 engineered to be secreted show premature differentiation of the entire lens epithelium. In contrast, transgenic mice engineered to secrete FGF-2 in the lens do not undergo premature differentiation of the lens epithelium (C. M. Stolen et al., 1997, Development 124, 4009-4017). To further assess the roles of FGFs and FGF receptors in lens development, the alpha A-crystallin promoter was used to target expression of FGF-3 to the developing lens of transgenic mice. The expression of FGF-3 in the lens rapidly induced epithelial cells throughout the lens to elongate and to express fiber cell-specific proteins including MIP and beta-crystallins. This premature differentiation of the lens epithelium was followed by the degeneration of the entire lens. Since FGF-1 and FGF-3 can both activate one FGF receptor isoform (FGFR2 IIIb) that is not activated by FGF-2, these results suggest that activation of FGFR2 IIIb is sufficient to induce fiber cell differentiation throughout the lens epithelium in vivo. Furthermore, transgenic lens cells expressing FGF-3 were able to induce the differentiation of neighboring nontransgenic lens epithelial cells in chimeric mice. Expression of FGF-3 in the lens also resulted in developmental alterations of the eyelids, cornea, and retina, and in the most severely affected transgenic lines, the postnatal appearance of intraocular glandular structures.
Animals↗