Fibroblast growth factor (FGF)-21 signals through both FGF receptor-1 and 2

Fibroblast growth factor (FGF)-21 is a member of the FGF superfamily based on sequence homology. However, unlike most members of this family it does not show any mitogenic activity in all cell types tested. The objective of this study is to identify and characterize receptors for this molecule. Sequencing of the cDNA clones from 3T3-L1 adipocytes indicates that the only isoforms for FGFR-1 and 2 expressed in 3T3-L1 cells are 1IIIc and 2IIIc, respectively, suggesting that FGF-21 regulates glucose metabolism in 3T3-L1 adipocytes through FGFR-1IIIc and FGFR-2IIIc.     

Fas-associated factor-1 inhibits nuclear factor-kappaB (NF-kappaB) activity by interfering with nuclear translocation of the RelA (p65) subunit of NF-kappaB

Fas-associated factor-1 (FAF1) is a Fas-binding pro-apoptotic protein that is a component of the death-inducing signaling complex in Fas-mediated apoptosis. Here, we show that FAF1 is involved in negative regulation of NF-kappaB activation. Overexpression of FAF1 decreased the basal level of NF-kappaB activity in 293 cells. NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, interleukin-1beta, and lipopolysaccharide was also inhibited by FAF1 overexpression. Moreover, FAF1 suppressed NF-kappaB activation induced by transducers of diverse NF-kappaB-activating signals such as TNF receptor-associated factor-2 and -6, MEKK1, and IkappaB kinase-beta as well as NF-kappaB p65, one of the end point molecules in the NF-kappaB activation pathway, suggesting that NF-kappaB p65 might be a target molecule upon which FAF1 acts. Subsequent study disclosed that FAF1 physically interacts with NF-kappaB p65 and that the binding domain of FAF1 is the death effector domain (DED)-interacting domain (amino acids 181-381), where DEDs of the Fas-associated death domain protein and caspase-8 interact. The NF-kappaB activity-modulating potential of FAF1 was also mapped to the DED-interacting domain. Finally, overexpression of FAF1 prevented translocation of NF-kappaB p65 into the nucleus and decreased its DNA-binding activity upon TNFalpha treatment. This study presents a novel function of FAF1, in addition to the previously known function as a component of the Fas death-inducing signaling complex, i.e. NF-kappaB activity suppressor by cytoplasmic retention of NF-kappaB p65 via physical interaction.     

Analysis of endogenous and exogenous nuclear translocation of fibroblast growth factor-1 in NIH 3T3 cells.

Nuclear localization of fibroblast growth factors (FGF) have been reported by many laboratories. We demonstrate here that FGF-1, the precursor for acidic FGF contains a putative nuclear translocation sequence (NTS) NYKKPKL, which is able to direct the expression of the bacterial beta galactosidase (beta gal) gene to the nucleus of transfected NIH 3T3 cells. However, this NTS is unable to target either FGF-1 itself or a FGF-1-beta gal fusion protein into the nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus. Indeed, when FGF-1 was fused to the NTS derived from the yeast histone 2B gene, the chimeric construct also failed to be transported into the nucleus either by itself or as a beta gal fusion protein. Interestingly, when 125I-FGF-1 was used to stimulate quiescent NIH 3T3 cells, a significant amount of internalized 125I-FGF-1 (approximately 10%) was found within the nucleus and the nuclear localization of FGF-1 through the exogenous pathway could be significantly reduced by suramin, an inhibitor of the interaction of FGF-1 with its receptor. These data suggest that while FGF-1 contains a NTS, nuclear translocation requires an exogenous and not an endogenous pathway.     

Eukaryotic translation initiation factor-6 enhances histamine and IL-2 production in mast cells

Eukaryotic translation initiation factor (eIF)-6 is known to be important in ribosome biogenesis. Previously, we have discovered that eIF-6 mRNA is induced in lung in a murine model of asthma. We also found that there was enhanced eIF-6 expression in mast cells stimulated with PMA plus calcium ionophore. Therefore, we hypothesized that the induction of eIF-6 enhances the production of bioactive mediators by mast cells upon allergic stimulation. In the current study, we found that eIF-6 mRNA was rapidly induced in murine mast cells stimulated by Fc epsilon RI cross-linking, which is a major physiologic stimulant for mast cells. eIF-6 was also induced in human mast cells upon stimulation. The increase in eIF-6 gene expression in murine mast cells was blocked by therapeutic agents such as dexamethasone and cyclosporin A. To determine the location and function of eIF-6, murine mast cells were transfected with a construct that overexpressed enhanced green fluorescent protein-tagged eIF-6. These experiments demonstrated that eIF-6 was localized predominantly in the nucleolus of the mast cells. Also, overexpression of enhanced green fluorescent protein/eIF-6 enhanced the production of histamine and IL-2, but not IL-4 by stimulated murine mast cells. These results suggest that eIF-6 regulates the production of selected bioactive mediators in allergic diseases. This is the first demonstration of a biologic function of eIF-6 in mammalian cells.     

The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic growth factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and is thought to play an important role in the mesoderm induction. Although hematopoietic cells derive from the mesoderm, relatively few studies have, until recently, addressed the role of FGF-2 in hematopoiesis. FGF-2 is expressed in cells of the bone marrow including stromal cells, and possibly cells from several hematopoietic cell lineages. It is stored in the bone marrow extra-cellular matrix and released by enzymes such as heparanase, plasmin, or phospholipase C and D. FGF-receptors (FGF-Rs) are expressed in leukemic cell lines and in hematopoietic cells. FGF-2 positively regulates hematopoiesis, by acting on stromal cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. The action of FGF-2 is most likely indirect since its action, on megakaryocytopoiesis for example, is abrogated by anti-IL6 antibodies. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of TGF-β Taken together, these results demonstrate that FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.     

FIF [fibroblast growth factor-2 (FGF-2)-interacting-factor], a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2

Numerous evidence indicates that some of the activities of fibroblast growth factor 2 (FGF-2) depend on an intracrine mode of action. Recently, we showed that three high molecular mass (HMM) nuclear forms of FGF-2 are part of a 320-kDa protein complex while the cytoplasmic AUG-initiated form is included in a 130-kDa complex. Consequently, the characterization of FGF endogenous targets has become crucial to allow the elucidation of their endogenous activities. Through the screening of GAL4-based yeast two-hybrid expression libraries, we have isolated a gene encoding a nuclear protein of 55 kDa, FIF (FGF-2-interacting-factor), which interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6. In this system, FIF interacts equally well with the NH2-extended 24-kDa FGF form as with the 18-kDa form, indicating that the FIF-binding motif is located in the last 155 amino acids of FGF-2. Nevertheless, coimmunoprecipitation experiments showed an exclusive association with HMM FGF-2. The predicted protein contains a canonical leucine zipper domain and three overlapping hydrophobic heptad repeats. The region spanning these repeats is, together with a region located in the N-terminal part of the FIF protein, implicated in the binding to FGF-2. In contrast to the full-length FIF protein, several deletion constructs were able to transactivate a lac-Z reporter gene. Furthermore, the COOH-terminal part, but not the full-length FIF protein, has previously been shown to exhibit antiapoptotic properties. Thus we discuss the possibility that these activities could reflect a physiological function of FIF through its interaction with FGF-2.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants. Not surprisingly, changes in O-GlcNAc protein modifications were noted during cell activation with and without GlcN addition. These findings could not be explained by some non-specific change in cell metabolism because ATP concentrations did not significantly change. We speculate that HBP-active compounds may contribute to patient care in certain inflammatory and autoimmune diseases.     

Costimulation of T cell receptor-triggered IL-2 production by Jurkat T cells via fibroblast growth factor receptor 1 upon its engagement by CD56

Recent studies have demonstrated that neural cell adhesion molecule (NCAM) is involved in multiple adhesive interactions with several different classes of ligands on the cell surface and in the extracellular matrix. One of these ligands is fibroblast growth factor receptor (FGFR) that is expressed on neural cells. While it is known that CD56 is a molecular isoform of NCAM expressed on human NK cells and a subset of T cells, it remains poorly characterized, with its ligand unidentified. Therefore, we were prompted to examine if CD56 molecules on NK cells interact with FGFR expressed on T cells. We demonstrate that ligation of FGFR1 beta on J.C2-14 Jurkat T cells by CD56 on fixed NK-92 cells costimulates TCR/CD3-triggered IL-2 production. CD56-binding mAbs inhibited the costimulatory effect of NK-92 cells in 50-75%. Flow cytometric analysis and cell adhesion assays showed that FGFR1 beta/Fc and FGFR2 beta/Fc chimeric proteins bind to NK-92 cells. The binding of FGFR1 beta/Fc protein to CD56 molecules was verified by immunoprecipitation of CD56 with anti-CD56 mAb followed by Western blotting with FGFR1 beta/Fc. These findings suggest that ligation of FGFR1 by CD56 may contribute to the interaction between NK cells and T cells that we have postulated in our previous studies.     

Fibroblast growth factor-12 (FGF12) translocation into intestinal epithelial cells is dependent on a novel cell-penetrating peptide domain: involvement of internalization in the in vivo role of exogenous FGF12

The extracellular effect of fibroblast growth factor-12 (FGF12) remains unknown because FGF12 cannot activate any fibroblast growth factor receptors (FGFRs), and FGF12 is not currently thought to be released from cells. We reported previously that FGF12 plays an intracellular role in the inhibition of radiation-induced apoptosis. In this study, we demonstrated that recombinant FGF12 was able to be internalized into the cytoplasm of a rat intestinal epithelial cell line, IEC6, and this process was dependent on two novel cell-penetrating peptide (CPP) domains (CPP-M and CPP-C). In particular, CPP-C, composed of ～10 amino acids, was identified as a specific domain of FGF12 and its subfamily in the C-terminal region (residues 140-149), although CPP-M was a common domain in the internal region of the FGF family. The absence of CPP-C from FGF12 or a mutation (E142L) in the CPP-C domain drastically reduced the internalization of FGF12 into cells. Therefore, CPP-C played an essential role in the internalization of FGF12. In addition, CPP-C was able to deliver other polypeptides into cells as a CPP because an FGF1/CPP-C chimeric protein was internalized into IEC6 cells more efficiently than wild-type FGF1. Finally, intraperitoneally added FGF12 inhibited radiation-induced apoptosis in the intestinal epithelial cells of BALB/c mice, and deletion of the CPP-C domain decreased the inhibition of the apoptosis. These findings suggest that exogenous FGF12 can play a role in tissues by translocating into cells through the plasma membrane, and the availability of this novel CPP provides a new tool for the intracellular delivery of bioactive molecules.     

Prostaglandins differently regulate FGF-2 and FGF receptor expression and induce nuclear translocation in osteoblasts via MAPK kinase

We have previously reported that prostaglandin F₂ (PGF₂) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF₂ or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF₂ stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF₂. Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF₂. Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF₂ exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF₂ stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.This research was supported by grants from University of Camerino and Fondazione Carima Italy and by National Institutes of Health Grant AR-46025 (to M.M.H)     

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2)

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.     

Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF- 2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2- treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.     

Age-dependent changes in Fibroblast growth factor 2 (FGF-2) expression in mouse cerebellar neurons

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor that regulates many neuronal functions and survival. We have characterised FGF-2 expression immunohistochemically in the cerebellum of young (4 months) and old (22 months) mice. About half of the population of the granule cells (GC), and all Purkinje cells (PC) expressed FGF-2 in all folia of the cerebellum at both ages. FGF-2 showed differential intracellular localization: predominantly localised to the nuclei of GC and present mainly in the cytosol of PC. There was a statistically significant (P = 0.0028) reduction in the number of FGF-2-positive GC in the cerebella of old (41.3+/-0.91%) compared to young (48.5+/-1.67%) mice, whereas no statistically significant age-dependent difference occurred in the number of FGF-2 positive PC. These results indicate a possible role of FGF-2 in cerebellar ageing.     

Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.     

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone-related matrix and plays multiple regulatory roles in mineralizing and non-mineralizing tissue. In osteogenic cell-lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)-2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF-2 decreased the mRNA expression of almost every bone-related protein in MPDL22, FGF-2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF-2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF-2-induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF-2-induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate-induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF-2 induces unique expression of osteopontin, which may play a role different from the other bone-related proteins during the process of periodontal tissue regeneration by FGF-2.     

Rapamycin antagonizes NF-kappaB nuclear translocation activated by TNF-alpha in primary vascular smooth muscle cells and enhances apoptosis

Several lines of evidence support the view that rapamycin inhibits NF-kappaB. TNF-alpha, a potent inducer of NF-kappaB, is released after artery injury (e.g., balloon angioplasty) and plays an important role in inflammation and restenosis. We investigated the effect of rapamycin on NF-kappaB activation and apoptosis in vascular smooth muscle cells (VSMCs) stimulated with TNF-alpha. Using EMSA, we found that TNF-alpha caused NF-kappaB nuclear translocation in VSMCs after 1 h of incubation. Rapamycin inhibited IkappaBalpha degradation, thereby preventing nuclear translocation. Activation of NF-kappaB was accompanied by an increase of Bcl-xL and Bfl-1/A1 proteins, detected by Western blot assay, whereas rapamycin prevented the TNF-alpha-induced enhancement of these antiapoptotic proteins. The extent of apoptosis of VSMCs exposed to TNF-alpha was significantly enhanced by rapamycin. The effect of rapamycin appeared to be independent of the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented IkappaBalpha degradation nor increased apoptosis of cells incubated with TNF-alpha. Finally, we demonstrate that the large immunophilin FK-506 binding protein FKBP51 is essential for TNF-alpha-induced NF-kappaB activation in VSMCs. Our findings show that rapamycin inhibits NF-kappaB activation and acts in concert with TNF-alpha in induction of VSMC apoptosis.