Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the 'variable' secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1beta receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG. (+info)
Expression of vascular endothelial growth factor in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat bladder carcinogenesis.
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Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are proteins implicated in tumor-associated microvascular angiogenesis. Expressions of VEGF and bFGF in various stages of chemical-induced rat bladder carcinogenesis were immunohistochemically investigated. Thirty-two male 6-week-old Wistar rats were given drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 20 weeks. VEGF and bFGF were not detected in the normal bladder epithelium. In simple hyperplasia, intensive expression of VEGF was observed in a few epithelial cells, and the expression of epithelial VEGF became more pronounced in papillary or nodular (PN) hyperplasia and papilloma. In carcinoma, heterogeneous expression of VEGF was observed in focal tumor cells, intensely expressed in the invading tumor cells. Ultrastructurally, carcinoma cells showed VEGF immunoreactivity in the cytoplasmic matrix and some rough endoplasmic reticulum, and VEGF-positive and -negative carcinoma cells were also clearly defined. High levels of VEGF mRNA were observed in the carcinoma. However, bFGF was not detected in the epithelium throughout the carcinogenesis. Increased microvessel counts appeared at simple hyperplasia and became more pronounced in PN hyperplasia, papilloma, and carcinoma (F-test; P < 0.05). In the carcinoma, the microvessel counts of the VEGF-expressing tumor areas were significantly higher than that of the non-VEGF-expressing tumor areas (U-test; P < 0.05). The present study suggests that upregulation of epithelial VEGF may begin at a quite early stage in BBN-induced rat bladder carcinogenesis, but bFGF may not be involved. (+info)
Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo.
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The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix. (+info)
Repression of human fibroblast growth factor 2 by a novel transcription factor.
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Here we describe the cloning of the regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) using a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence. Overexpression of human RFT (RFT-A) reduces FGF-2 RNA and protein levels in both normal and tumor cell lines. Its splice variants, RFT-A' and RFT-B, have deletions in the putative DNA binding domain and fail to bind FGF-2 promoter and repress FGF-2 gene expression. The ratios of RFT isoforms differ between normal and tumor cells, with the splice variants dominating in tumor cells. Overexpression of RFT-A induces glioma cell death. Our data suggest that regulation of FGF-2 by RFT is important for cellular functions and may be impaired in certain tumors. (+info)
Stimulation of tumour growth by wound-derived growth factors.
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The goal of this work was to determine the molecular basis for the induction of tumour vascularization and progression by injury. Magnetic resonance imaging (MRI) studies demonstrated that administration of wound fluid derived from cutaneous injuries in pigs reduced the lag for vascularization and initiation of growth of C6 glioma spheroids, implanted in nude mice, and accelerated tumour doubling time. The former effect can be attributed to the angiogenic capacity of wound fluid as detected in vivo by MRI, and in vitro in promoting endothelial cell proliferation. The latter effect, namely the induced rate of tumour growth, is consistent with the angiogenic activity of wound fluid as well as with the finding that wound fluid was directly mitogenic to the tumour cells, and accelerated growth of C6 glioma in spheroid culture. Of the multiple growth factors present in wound fluid, two key factors, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and platelet-derived growth factor (PDGF), were identified as the dominant mitogens for C6 glioma, and inhibition of their activity using specific neutralizing antibodies suppressed the mitogenic effect of wound fluid on DNA synthesis in C6 glioma. This study suggests that the stimulatory effect of injury on tumour progression can possibly be attenuated by therapeutic targeting directed against a limited number of specific growth factors. (+info)
Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors.
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Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner. (+info)
Growth factors prevent changes in Bcl-2 and Bax expression and neuronal apoptosis induced by nitric oxide.
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Recent studies have shown that nitric oxide (NO) donors can trigger apoptosis of neurons, and growth factors such as insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) can protect against NO-induced neuronal cell death. The purpose of this study was to elucidate the possible mechanisms of NO-mediated neuronal apoptosis and the neuroprotective action of these growth factors. Both IGF-1 and bFGF prevented apoptosis induced by NO donors, sodium nitroprusside (SNP) or 3-morpholinosydnonimin (SIN-1) in hippocampal neuronal cultures. Incubation of neurons with SNP induced caspase-3-like activation following downregulation of Bcl-2 and upregulation of Bax protein levels in cultured neurons. Treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by SNP. In addition, treatment of neurons with an inhibitor of caspase-3, Ac-DEVD-CHO, together with SNP did not affect the changes in the protein levels, although it inhibited NO-induced cell death. Pretreatment of cultures with either IGF-1 or bFGF prior to NO exposure inhibited caspase-3-like activation together with the changes in Bcl-2 and Bax protein levels. These results suggest that the changes in Bcl-2 and Bax protein levels followed by caspase-3-like activation are a component in the cascade of NO-induced neuronal apoptosis, and that the neuroprotective actions of IGF-1 and bFGF might be due to inhibition of the changes in the protein levels of the Bcl-2 family. (+info)
Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism.
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In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis. (+info)