Direct interaction between CCN family protein 2 and fibroblast growth factor 1

In an attempt to find out a new molecular counterpart of CCN family protein 2 (CCN2), a matricellular protein with multiple functions, we performed an interactome analysis and found fibroblast growth factor (FGF) -1 as one of the candidates. Solid-phase binding assay indicated specific binding between CCN2 and FGF-1. This binding was also confirmed by surface plasmon resonance (SPR) analysis that revealed a dissociation constant (Kd) of 3.98 nM indicating strong molecular interaction between the two. RNA analysis suggested that both FGF-1 and CCN2 could be produced by chondrocytes and thus their interaction in the cartilage is possible. These findings for the first time indicate the direct interaction of CCN2 and FGF-1 and suggest the co-presence of these molecules in the cartilage microenvironment. CCN2 is a well-known promoter of cartilage development and regeneration, whereas the physiological and pathological role of FGF-1 in cartilage mostly remains unclear. Biological role of FGF-1 itself in cartilage is also suspected.     

Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis

Two members of the fibroblast growth factor (FGF) family, basic FGF (bFGF) and FGF-18, have been implicated in the regulation of articular and intervertebral disc (IVD) cartilage homeostasis. Studies on bFGF from a variety of species have yielded contradictory results with regards to its precise role in cartilage matrix synthesis and degradation. In contrast, FGF-18 is a well-known anabolic growth factor involved in chondrogenesis and articular cartilage repair. In this review, we examined the biological actions of bFGF and FGF-18 in articular and IVD cartilage, the specific cell surface receptors bound by each factor, and the unique signaling cascades and molecular pathways utilized to exert their biological effects. Evidence suggests that bFGF selectively activates FGF receptor 1 (FGFR1) to exert degradative effects in both human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme activity, inhibition of matrix production, and increased cell proliferation resulting in clustering of cells seen in arthritic states. FGF-18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating FGFR3, increasing matrix formation and cell differentiation while inhibiting cell proliferation, leading to dispersed cells surrounded by abundant matrix. The results from in vitro and in vivo studies suggest the potential usefulness of bFGF and FGFR1 antagonists, as well as FGF-18 and FGFR3 agonists, as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future.     

Expression of fibroblast growth factor family during postnatal skeletal muscle hypertrophy

The potential role of the fibroblast growth factor (FGF) familyduring stretch-induced postnatal skeletal muscle hypertrophy wasanalyzed by using an avian wing-weighting model. After 2 or 11 days ofweighted stretch, anterior latissimus dorsi (ALD) muscles were, onaverage, 34 (P < 0.01) and 85%(P < 0.01) larger, respectively, than unweighted ALD control muscles. By using quantitative RT-PCR, FGF-1 mRNA expression was found to be significantly decreased in ALDmuscles stretched for 2 or 11 days. In contrast, FGF-4 and FGF-10 mRNAexpression was significantly increased 2 days after initiation ofstretch. FGF-2, FGF-10, fibroblast growth factor receptor 1, andFREK mRNA expression was significantly increased at 11 days poststretch. Increases in FGF-2 and FGF-4 protein could bedetected throughout the myofiber periphery after 11 days of stretch. Ona cellular level, FGF-2 and FGF-4 proteins were differentiallylocalized. This differential expression pattern and proteinlocalization of the FGF family in response to stretch-induced hypertrophy suggest distinct roles for individual FGFs during thepostnatal hypertrophy process.

     

Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors.

Ligand-induced dimerization and transphosphorylation are thought to be important events by which receptor tyrosine kinases generate cellular signals. We have investigated the ability of signalling-defective, truncated fibroblast growth factor (FGF) receptors (FGFR-1 and FGFR-2) to block the FGF response in cells that express both types of endogenous FGF receptors. When these dominant negative receptors are expressed in NIH 3T3 cells transformed by the secreted FGF-4, the transformed properties of the cells can be reverted to various degrees, with better reversion phenotype correlating with higher levels of truncated receptor expression. Furthermore, truncated FGFR-2 is significantly more efficient at producing reversion than FGFR-1, indicating that FGF-4 preferentially utilizes the FGFR-2 signalling pathway. NIH 3T3 clones expressing these truncated receptors are more resistant to FGF-induced mitogenesis and also exhibit reduced tyrosine phosphorylation upon treatment with FGF. The block in FGF-signalling, however, can be overcome by the addition of excess growth factor. The truncated receptors have binding affinities that are four- to eightfold lower than those of wild-type receptors, as measured by Scatchard analysis. We also observed a partial specificity in the responses of truncated-receptor-expressing clones to FGF-2 or FGF-4. Our results suggest that the block to signal transduction produced by kinase-negative FGF receptors is achieved through a combination of dominant negative effects and competition for growth factor binding with functional receptors.     

The fibroblast growth factor family: Structural and biological properties

This article sumarizes the structural and biological properties of the family of fibroblast growth factors (FGF). Basic FGF (bFGF) and acidic FGF (aFGF) are the best characterized members of this family. bFGF and aFGF are potent modulators of cell proliferation, motility and differentiation. They are also potent angiogenesis factors in vivo. Some of the important biological characteristics of bFGF and aFGF discussed in the review include the affinity of bFGF and aFGF for heparin, their lack of secretion in culture and their association with extracellular matrix. Recently, several oncogenes, 40–50% homologous in sequence to bFGF and aFGF have been identified. These include int-2, hst, K-fgf and FGF-5. The structural and biological properties of these FGF-related oncogenes are also discussed.     

Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex

The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.     

FGF-19, a novel fibroblast growth factor with unique specificity for FGFR4.

We have identified a novel fibroblast growth factor, FGF-19, the most distant member of the FGF family described to date. FGF-19 is a high affinity, heparin dependent ligand for FGFR4 and is the first member of the FGF family to show exclusive binding to FGFR4. Human FGF-19 maps to chromosome 11 q13.1, a region associated with an osteoporosis-pseudoglioma syndrome of skeletal and retinal defects. FGF-19 message is expressed in several tissues including fetal cartilage, skin, and retina, as well as adult gall bladder and is overexpressed in a colon adenocarcinoma cell line.     

Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.     

New culture system for human embryonic stem cells: autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP₃) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of G_i-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with G_i-proteins. Pretreatment of acini with pertussis toxin which inhibits G_i-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP₃ production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and G_i-type G-proteins and that G_i-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.     

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.     

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.