Faculty Bibliography

HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs

Fibroblast growth factors (FGFs) are key regulators of several developmental processes in which cell fate and differentiation to various tissue lineages are determined. The importance of the proper spatial and temporal regulation of FGF signals is evident from human and mouse genetic studies which show that mutations leading to the dysregulation of FGF signals cause a variety of developmental disorders including dominant skeletal diseases and cancer. The FGF ligands signal via a family of receptor tyrosine kinases and, depending on the cell type or stage of maturation, produce diverse biological responses that include proliferation, growth arrest, differentiation or apoptosis. A central issue in FGF biology is to understand how these diverse cellular responses are determined and how similar signaling inputs can generate distinct patterns of gene expression that govern the specificity of the cellular response. In this review we draw upon studies from the past fifteen years and attempt to construct a molecular picture of the different levels of regulation by which such specific cellular responses could be achieved by FGF signals. We discuss whether specificity could lie in the nature of the ligand, the particular receptor, the signal transduction pathways utilized, or the transcriptional regulation of specific genes. Finally, we also discuss how the interplay of FGF signals with other signaling systems could contribute to the cellular response. In particular we focus on the interaction with the Wnt pathway since FGF/Wnt cross-talk is emerging as an important nexus in regulating a variety of biological processes

Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway

FGF4 is the earliest member of the fibroblast growth factor (FGF) family expressed during embryogenesis where it plays essential roles in post-implantation development and limb growth and patterning. The expression of the Fgf4 gene in specific developmental stages, including the ICM of the blastocyst, the myotomes, and the limb bud AER, is regulated by distinct enhancer elements (Hom) in the 3' UTR. We previously identified the Hom3a region as the major DNA element responsible for Fgf4 expression in the myotomes and AER, and showed that a conserved E-box is a target for the myogenic bHLH transcription factors MYF5 and MYOD. To further define the cis- and trans-acting elements that determine Hom3a activity, we conducted a mutational analysis of the ability of the Hom3a region to drive lacZ expression in the myotomes of transgenic mice. We identified a minimal enhancer of 226nt that contains four elements, including the E-box, necessary to drive gene expression in the myotomes. One of these elements is a binding site for the GATA family of transcription factors, and we show here that GATA 1-4 and 6 can synergize with MYF5 or MYOD to activate transcription of a reporter plasmid driven by a portion of the Hom3a enhancer including the GATA site and the E-box. In line with this finding, we could show a direct interaction between MYF5/MYOD and GATA-3 or GATA-4, mediated by the N-terminal and bHLH domains of MYF5/MYOD and the C-terminal zing finger domain of GATA-3/4. To further study the role of the Hom3a enhancer in directing Fgf4 expression and the function of FGF4 in limb and muscle development, we generated mutant mice in which the Fgf4 Hom3a region had been deleted (Delta3a). In situ hybridization analysis of sections from Delta3a/ Delta3a embryos at E11.5 showed a drastically reduced expression of Fgf4 mRNA in the myotomes and AER. However, these mice developed normally and show no limb or muscle defects, and the same was true of heterozygous mice in which one Fgf4 allele carried the Hom3a deletion and the other was a null allele (Delta3a/Fgf4(-)). Together, these results show that Hom3a is the major DNA enhancer element directing Fgf4 expression in myotomes and limb bud AER, and that its activity in the myotomes results at least in part from the synergistic action of GATA and bHLH myogenic factors that bind to evolutionary conserved sequences in the Hom3a enhancer. However, expression of Fgf4 in the myotomes or AER of murine embryos does not appear to be essential for muscle or limb development

The prototypical fibroblast growth factor receptor (FGFR) extracellular domain consists of three Ig domains (D1-D3) of which the two membrane-proximal D2 and D3 domains and the interconnecting D2-D3 linker bear the determinants of ligand binding and specificity. In contrast, D1 and the D1-D2 linker are thought to play autoinhibitory roles in FGFR regulation. Here, we report the crystal structure of the three-Ig form of FGFR3c in complex with FGF1, an FGF that binds promiscuously to each of the seven principal FGFRs. In this structure, D1 and the D1-D2 linker are completely disordered, demonstrating that these regions are dispensable for FGF binding. Real-time binding experiments using surface plasmon resonance show that relative to two-Ig form, the three-Ig form of FGFR3c exhibits lower affinity for both FGF1 and heparin. Importantly, we demonstrate that this autoinhibition is mediated by intramolecular interactions of D1 and the D1-D2 linker with the minimal FGF and heparin-binding D2-D3 region. As in the FGF1-FGFR2c structure, but not the FGF1-FGFR1c structure, the alternatively spliced betaC'-betaE loop is ordered and interacts with FGF1 in the FGF1-FGFR3c structure. However, in contrast to the FGF1-FGFR2c structure in which the betaC'-betaE loop interacts with the beta-trefoil core region of FGF1, in the FGF1-FGFR3c structure, this loop interacts extensively with the N-terminal region of FGF1, underscoring the importance of the FGF1 N terminus in conferring receptor-binding affinity and promiscuity. Importantly, comparison of the three FGF1-FGFR structures shows that the flexibility of the betaC'-betaE loop is a major determinant of ligand-binding specificity and promiscuity

Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition

Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis

Fibroblast growth factor (FGF) 2 (or basic FGF) is expressed at increased levels in human prostate cancer. FGF2 can promote cell motility and proliferation, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGF2 plays a critical role in prostate cancer progression, we have used the transgenic adenocarcinoma of the mouse prostate (TRAMP) model system. A high percentage of TRAMP mice develop metastatic prostate cancer, and thus the TRAMP model is useful for evaluating cancer progression. TRAMP mice were crossed with FGF2 knockout (FGF2(-/-)) mice, and tumor progression in TRAMP mice that were either hemi- or homozygous for inactivation of the FGF2 allele was compared with progression in wild-type TRAMP mice. Inactivation of even one FGF2 allele resulted in increased survival, a decrease in metastasis, and inhibition of progression to the poorly differentiated phenotype in primary prostatic tumors. When compared with wild-type mice, poorly differentiated tumors arising in FGF(+/-) and FGF(-/-) mice expressed higher levels of vascular endothelial growth factor and, in some cases, increased levels of acidic FGF intracellular binding protein, a nuclear FGF1-binding protein. These findings suggest that both FGF2-mediated angiogenesis and intranuclear FGF2 activities may promote tumor progression and support the hypothesis that FGF2 plays a significant role in prostate cancer progression in vivo

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate