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Moosa Mohammadi - One of the best experts on this subject based on the ideXlab platform.
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Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology
Cold Spring Harbor Perspectives in Biology, 2013Co-Authors: Artur A. Belov, Moosa MohammadiAbstract:Fibroblast growth factor (FGF) signaling fulfills essential roles in metazoan development and metabolism. A wealth of literature has documented the requirement for FGF signaling in multiple processes during embryogenesis, including implantation (Feldman et al. 1995), gastrulation (Sun et al. 1999), somitogenesis (Dubrulle and Pourquie 2004; Wahl et al. 2007; Lee et al. 2009; Naiche et al. 2011; Niwa et al. 2011), body plan formation (Martin 1998; Rodriguez Esteban et al. 1999; Tanaka et al. 2005; Mariani et al. 2008), morphogenesis (Metzger et al. 2008; Makarenkova et al. 2009), and organogenesis (Goldfarb 1996; Kato and Sekine 1999; Sekine et al. 1999; Sun et al. 1999; Colvin et al. 2001; Serls et al. 2005; Vega-Hernandez et al. 2011). Recent clinical and biochemical data have uncovered unexpected roles for FGF signaling in metabolic processes, including phosphate/vitamin D homeostasis (Consortium 2000; Razzaque and Lanske 2007; Nakatani et al. 2009; Gattineni et al. 2011; Kir et al. 2011), cholesterol/bile acid homeostasis (Yu et al. 2000a; Holt et al. 2003), and glucose/lipid metabolism (Fu et al. 2004; Moyers et al. 2007). Highlighting its diverse biology, deranged FGF signaling contributes to many human diseases, such as congenital craniosynostosis and dwarfism syndromes (Naski et al. 1996; Wilkie et al. 2002, 2005), Kallmann syndrome (Dode et al. 2003; Pitteloud et al. 2006a), hearing loss (Tekin et al. 2007, 2008), and renal phosphate wasting disorders (Shimada et al. 2001; White et al. 2001), as well as many acquired forms of cancers (Rand et al. 2005; Pollock et al. 2007; Gartside et al. 2009; di Martino et al. 2012). Endocrine FGFs have also been implicated in the progression of acquired metabolic disorders, including chronic kidney disease (Fliser et al. 2007), obesity (Inagaki et al. 2007; Moyers et al. 2007; Reinehr et al. 2012), and insulin resistance (Fu et al. 2004; Chen et al. 2008b; Chateau et al. 2010; Huang et al. 2011), giving rise to many opportunities for drug discovery in the field of FGF biology (Beenken and Mohammadi 2012). Based on sequence homology and phylogeny, the 18 mammalian FGFs are grouped into six subfamilies (Ornitz and Itoh 2001; Popovici et al. 2005; Itoh and Ornitz 2011). Five of these subfamilies act in a paracrine fashion, namely, the FGF1 subfamily (FGF1 and FGF2), the FGF4 subfamily (FGF4, FGF5, and FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, and FGF22), the FGF8 subfamily (FGF8, FGF17, and FGF18), and the FGF9 subfamily (FGF9, FGF16, and FGF20). In contrast, the FGF19 subfamily (FGF19, FGF21, and FGF23) signals in an endocrine manner (Beenken and Mohammadi 2012). FGFs exert their pleiotropic effects by binding and activating the FGF receptor (FGFR) subfamily of receptor tyrosine kinases that are coded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) in mammals (Johnson and Williams 1993; Mohammadi et al. 2005b). The extracellular domain of FGFRs consists of three immunoglobulin (Ig)-like domains (D1, D2, and D3), and the intracellular domain harbors the conserved tyrosine kinase domain flanked by the flexible amino-terminal juxtamembrane linker and carboxy-terminal tail (Lee et al. 1989; Dionne et al. 1991; Givol and Yayon 1992). A unique feature of FGFRs is the presence of a contiguous segment of glutamic and aspartic acids in the D1–D2 linker, termed the acid box (AB). The two-membrane proximal D2 and D3 and the intervening D2–D3 linker are necessary and sufficient for ligand binding/specificity (Dionne et al. 1990; Johnson et al. 1990), whereas D1 and the D1–D2 linker are implicated in receptor autoinhibition (Wang et al. 1995; Roghani and Moscatelli 2007; Kalinina et al. 2012). Alternative splicing and translational initiation further diversify both ligands and receptors. The amino-terminal regions of FGF8 and FGF17 can be differentially spliced to yield FGF8a, FGF8b, FGF8e, FGF8f (Gemel et al. 1996; Blunt et al. 1997), and FGF17a and FGF17b isoforms (Xu et al. 1999), whereas cytosine-thymine-guanine (CTG)-mediated translational initiation gives rise to multiple high molecular weight isoforms of FGF2 and FGF3 (Florkiewicz and Sommer 1989; Prats et al. 1989; Acland et al. 1990). The tissue-specific alternative splicing in D3 of FGFR1, FGFR2, and FGFR3 yields “b” and “c” receptor isoforms which, along with their temporal and spatial expression patterns, is the major regulator of FGF–FGFR specificity/promiscuity (Orr-Urtreger et al. 1993; Ornitz et al. 1996; Zhang et al. 2006). A large body of structural data on FGF–FGFR complexes has begun to reveal the intricate mechanisms by which different FGFs and FGFRs combine selectively to generate quantitatively and qualitatively different intracellular signals, culminating in distinct biological responses. In addition, these structural data have unveiled how pathogenic mutations hijack the normal physiological mechanisms of FGFR regulation to lead to pathogenesis. We will discuss the current state of the structural biology of the FGF–FGFR system, lessons learned from studying the mechanism of action of pathogenic mutations, and how the structural data are beginning to shape and advance the translational research.
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plasticity in interactions of fibroblast growth factor 1 fgf1 n terminus with fgf receptors underlies promiscuity of fgf1
Journal of Biological Chemistry, 2012Co-Authors: Andrew Beenken, Anna V Eliseenkova, Omar A Ibrahimi, Shaun K Olsen, Moosa MohammadiAbstract:Abstract Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1–3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the “universal FGFR ligand” because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the “b” and “c” splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 A resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.
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The FGF family: biology, pathophysiology and therapy
Nature Reviews Drug Discovery, 2009Co-Authors: Andrew Beenken, Moosa MohammadiAbstract:Fibroblast growth factors (FGFs) signal through FGF receptor tyrosine kinases to regulate a wide range of biological processes during development and adulthood. FGF receptors (FGFRs) are involved in the pathogenesis of cancer and skeletal disorders. Experiments in model systems have shown that FGFR-specific inhibitors may be valuable in treating multiple myeloma, bladder and endometrial cancers. FGF1, FGF2 and FGF4 have been studied in clinical trials for the treatment of cardiovascular disease. The results of most of these trials have been unclear. However, some promising prospects remain. In particular, plasmids encoding FGF1 have shown potential in treating peripheral ischaemia. Recombinant FGF7 is Food and Drug Administration approved for the treatment of chemoradiation-induced oral mucositis. Research into the application of FGF7 to treat conditions such as graft-versus-host disease is ongoing. FGF18 increases cartilage formation in rats and may be useful in treating osteoarthritis. The endocrine FGF19 subfamily holds the greatest promise for therapeutic development. These ligands circulate throughout the body owing to weak affinity for HSGAG (heparan sulphate glycosaminoglycan), and they require α-klotho–β-klotho proteins as cofactors for their activity. The α-klotho–β-klotho proteins also determine the target-tissue specificity of FGF19 subfamily ligands. FGF19 negatively regulates bile acid synthesis and recombinant FGF19 increases the metabolic rate of mice. Unfortunately, FGF19 transgenic mice develop hepatocellular carcinomas, and this side effect may impede the pharmaceutical development of FGF19. FGF21 is a mediator of the fasting response that increases glucose uptake, improves insulin sensitivity and reduces serum glucagon and triglyceride levels. In contrast to FGF19, FGF21 is not mitogenic, and FGF21 administration leads to neither oedema nor hypoglycaemia, which are two common side effects of agents that modulate metabolic disorders. FGF21 thus shows great potential for treating type 2 diabetes. FGF23 reduces renal phosphate reabsorption and downregulates vitamin D activation. FGF23 is broadly implicated in human disease, including autosomal-dominant hypophosphataemic rickets, tumour-induced osteomalacia, familial tumoral calcinosis and end-stage kidney disease. Neutralizing antibodies against FGF23 have shown efficacy in model systems and demonstrate the clinical potential of FGF23-specific therapies. The family of fibroblast growth factors (FGFs) regulates a plethora of developmental processes, including brain patterning, branching morphogenesis and limb development. Several mitogenic, cytoprotective and angiogenic therapeutic applications of FGFs are already being explored, and the recent discovery of the crucial roles of the endocrine-acting FGF19 subfamily in bile acid, glucose and phosphate homeostasis has sparked renewed interest in the pharmacological potential of this family. This Review discusses traditional applications of recombinant FGFs and small-molecule FGF receptor kinase inhibitors in the treatment of cancer and cardiovascular disease and their emerging potential in the treatment of metabolic syndrome and hypophosphataemic diseases. The central roles of members of the fibroblast growth factor (FGF) family in human developmental processes and disease have been well documented. In their Review, Beenken and Mohammadi discuss the more established applications of FGF-related therapies in the treatment of cancer and cardiovascular disease, emphasizing their potential to modulate aspects of the metabolic syndrome and hypophosphataemic diseases.
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impaired fgf signaling contributes to cleft lip and palate
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Bridget M Riley, Moosa Mohammadi, Adela M Mansilla, Sandra Daackhirsch, Brion S Maher, Lisa M Raffensperger, Erilynn T Russo, A R Vieira, Catherine Dode, Mary L MarazitaAbstract:Nonsyndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from a combination of genetic and environmental factors. Several members of the FGF and FGFR families are expressed during craniofacial development and can rarely harbor mutations that result in human clefting syndromes. We hypothesized that disruptions in this pathway might also contribute to NS CLP. We sequenced the coding regions and performed association testing on 12 genes (FGFR1, FGFR2, FGFR3, FGF2, FGF3, FGF4, FGF7, FGF8, FGF9, FGF10, FGF18, and NUDT6) and used protein structure analyses to predict the function of amino acid variants. Seven likely disease-causing mutations were identified, including: one nonsense mutation (R609X) in FGFR1, a de novo missense mutation (D73H) in FGF8, and other missense variants in FGFR1, FGFR2, and FGFR3. Structural analysis of FGFR1, FGFR2, and FGF8 variants suggests that these mutations would impair the function of the proteins, albeit through different mechanisms. Genotyping of SNPs in the genes found associations between NS CLP and SNPs in FGF3, FGF7, FGF10, FGF18, and FGFR1. The data suggest that the FGF signaling pathway may contribute to as much as 3–5% of NS CLP and will be a consideration in the clinical management of CLP.
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fibroblast growth factor fgf homologous factors share structural but not functional homology with fgfs
Journal of Biological Chemistry, 2003Co-Authors: Shaun K Olsen, Anna V Eliseenkova, Mitchell Goldfarb, Meirav Garbi, Niccolo Zampieri, David M Ornitz, Moosa MohammadiAbstract:Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1–FHF4; also known as FGF11–FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a -trefoil fold consisting of 12 antiparallel strands (1 through 12). The FHF1b -trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structurebased mutational analysis, we identified two surface residues, Arg 52 in the 4–5 loop and Val 95 in the 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets.
James A Thomson - One of the best experts on this subject based on the ideXlab platform.
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thermal stability of fibroblast growth factor protein is a determinant factor in regulating self renewal differentiation and reprogramming in human pluripotent stem cells
Stem Cells, 2012Co-Authors: Guokai Chen, Daniel R Gulbranson, Zhonggang Hou, James A ThomsonAbstract:Fibroblast growth factor (FGF), transforming growth factor (TGF)/Nodal, and Insulin/insulin-like growth factor (IGF) signaling pathways are sufficient to maintain human embryonic stem cells (ESCs) and induced pluripotent stem cells in a proliferative, undifferentiated state. Here, we show that only a few FGF family members (FGF2, FGF4, FGF6, and FGF9) are able to sustain strong extracellular-signal-regulated kinase (ERK) phosphorylation and NANOG expression levels in human ESCs. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild-type protein, not receptor specificity, and that a mutated thermal-stable FGF1 sustains human ESCs and supports both differentiation and reprogramming protocols.
Malgorzata Zakrzewska - One of the best experts on this subject based on the ideXlab platform.
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Translocation of Exogenous FGF1 and FGF2 Protects the Cell against Apoptosis Independently of Receptor Activation.
Journal of molecular biology, 2018Co-Authors: Michal Janusz Kostas, Agata Lampart, Joanna Bober, Antoni Wiedlocha, Justyna Tomala, Daniel Krowarsch, Jacek Otlewski, Malgorzata ZakrzewskaAbstract:FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.
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Instability restricts signaling of multiple fibroblast growth factors
Cellular and Molecular Life Sciences, 2015Co-Authors: Marcela Buchtová, Radka Chaloupkova, Malgorzata Zakrzewska, Iva Vesela, Jana Barathova, Iva Gudernova, Renata Zajickova, Lukas Trantirek, Petra Celá, Jorge MartinAbstract:Fibroblast growth factors (FGFs) deliver extracellular signals that govern many developmental and regenerative processes, but the mechanisms regulating FGF signaling remain incompletely understood. Here, we explored the relationship between intrinsic stability of FGF proteins and their biological activity for all 18 members of the FGF family. We report that FGF1, FGF3, FGF4, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF20, and FGF22 exist as unstable proteins, which are rapidly degraded in cell cultivation media. Biological activity of FGF1, FGF3, FGF4, FGF6, FGF8, FGF10, FGF16, FGF17, and FGF20 is limited by their instability, manifesting as failure to activate FGF receptor signal transduction over long periods of time, and influence specific cell behavior in vitro and in vivo. Stabilization via exogenous heparin binding, introduction of stabilizing mutations or lowering the cell cultivation temperature rescues signaling of unstable FGFs. Thus, the intrinsic ligand instability is an important elementary level of regulation in the FGF signaling system.
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phosphorylation of fibroblast growth factor fgf receptor 1 at ser777 by p38 mitogen activated protein kinase regulates translocation of exogenous fgf1 to the cytosol and nucleus
Molecular and Cellular Biology, 2008Co-Authors: Vigdis Sorensen, Malgorzata Zakrzewska, Yan Zhen, Ellen Margrethe Haugsten, Sebastien Walchli, Trine Nilsen, Sjur Olsnes, Antoni G WiedlochaAbstract:Fibroblast growth factor 1 (FGF1) belongs to a family of heparin binding polypeptide growth factors encoded by 22 genes in mice and humans (20). Most FGFs transmit signals to cells by binding and through activation of a family of high-affinity, tyrosine kinase FGF receptors (FGFR1 to -4) (7). FGF1 and FGF2 may, in addition, be translocated from the extracellular space into the cytosol and nucleus of target cells (37, 39, 46, 58). Translocated FGF1 and FGF2, in particular nuclear FGF1 and FGF2, have been reported to be involved in regulating processes such as rRNA synthesis and cell growth (17-19, 21, 36, 44, 45, 52, 54, 56, 61). The translocation of exogenous FGF1 or FGF2 into the cytosol and nucleus is a highly regulated process that requires phosphatidylinositol 3-kinase (PI3K) activity (23) and active hsp90 (52) and is strictly dependent on binding of FGF to either FGFR1 or FGFR4 (47). Furthermore, translocation was found to be cell cycle dependent (3, 31, 63), it can be stimulated by serum deprivation of cells (1, 3, 18, 25, 31, 32, 55, 63), and it occurs after a several-hour delay compared to the endocytic uptake of FGF (31, 47). The nuclear trafficking of FGF1 is also tightly regulated by two nuclear localization sequences (19, 51), a nuclear export sequence (36), and by phosphorylation of FGF1 at Ser130 by protein kinase Cδ (PKCδ) (57). The actual translocation of FGF across cellular membranes appears to occur in early endosomes, as it was found to depend on the electrical potential across vesicular membranes (31, 32). Extensive unfolding of the growth factor is not required for the translocation to occur (53). It is not known exactly how FGF crosses the vesicular membrane and to what extent this event involves accessory proteins in addition to FGFR. Previously, we identified certain amino acid residues localized in the C-terminal tail region of FGFR that are crucial for the FGF1 translocation (47). p38 mitogen-activated protein kinase (MAPK) is a conserved member of the MAPK family and participates in a variety of biological processes. Originally, it was described as a kinase mainly involved in inflammatory and stress-induced responses, often activating proapoptotic stimuli, but it is also involved in regulating growth and differentiation of cells in response to a wide range of growth factors and cytokines (42, 62). More recently it has been shown that p38 MAPK signaling also functions in tumor suppression (15), and it can regulate endocytosis and intracellular sorting. p38 MAPK contributes to regulation of endocytosis by phosphorylation of components of the endocytic machinery, such as GDI-Rab5 (5), EEA1 (8, 28), and rabenosyn 5 (28). Furthermore, it was shown that p38 MAPK can regulate epidermal growth factor receptor (EGFR) internalization (49, 59, 65) and downregulation (9). Four isoforms of p38 MAPK have been identified in mammals: p38α, -β, -γ, and -δ. p38α and p38β are ubiquitously expressed, whereas p38γ is most prominent in muscle and p38δ is most prominent in lungs and kidneys. p38 MAPK is activated through the sequential activation of MAPK kinase kinases and MAPK kinases (MKKs). MKK3 and MKK6 are known to directly activate p38 MAPK by phosphorylating a Thr-Gly-Tyr dual phosphorylation motif in the activation loop of the p38 kinase subdomain VIII. It has also been shown that the p38α isoform can be activated by alternative mechanisms which involve stimulation of p38α autophosphorylation of its dual phosphorylation motif through interaction with TAB1 (12, 13, 22) or Zap 70 (43). Active p38 MAPK phosphorylates target substrates on serines and threonines, in particular its downstream substrate, MK2/MAPKAP kinase 2, which further activates various substrates, including HSP27, CREB, transcriptional factor ATF1, SRF, and eukaryotic initiation factor 4E (42, 62). p38 MAPK can be activated by FGF/FGFR and has been shown to be a crucial second messenger in regulation of various cellular responses to FGF, such as proliferation, migration, and growth inhibition (2, 11, 27, 29, 30, 34, 40). In the present study we investigated the role of p38 MAPK in the translocation of exogenous FGF1 into the cytosol and nucleus of cells. We demonstrate that FGF1 translocation requires activity of the α isoform of p38 MAPK and that it can be enhanced upon stimulation of the p38 MAPK activity by chemical stress. We also show that phosphorylation of FGFR1 at Ser777 is required for the translocation of FGF1 and that this phosphorylation is regulated by p38α.
Hyunmo Ryoo - One of the best experts on this subject based on the ideXlab platform.
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the protein kinase c pathway plays a central role in the fibroblast growth factor stimulated expression and transactivation activity of runx2
Journal of Biological Chemistry, 2003Co-Authors: Je-yong Choi, Hyunmo RyooAbstract:Abstract Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling induces the expression of Runx2, a key transcription factor in osteoblast differentiation, but little is known about the molecular signaling mechanisms that mediate this. Here we examined the role of the protein kinase C (PKC) pathway in regulating Runx2 gene expression and its transactivation function. Treatment with FGF2 or FGF4, or transfection with a vector expressing a mutant FGFR2 that is constitutively activated in the absence of ligand, strongly stimulates Runx2 expression. Electrophoretic mobility shift assays also showed that FGF2 treatment increases the specific binding of Runx2 to the cognate response element in the osteocalcin gene promoter. Blocking PKC completely inhibited FGF2-induced Runx2 expression, whereas mitogen-activate protein kinase inhibitors had no effect. The FGF/FGFR-stimulated 6xOSE2 promoter activity was also blocked by inhibiting PKC, as was the FGF2 stimulation of the DNA-binding activity of Runx2. Experiments with PKC isoform-specific inhibitors and dominant negative isoforms of PKC indicate that PKCδ is one of key isoforms involved in the FGF2-stimulated Runx2 expression. In addition, experiments with Runx2-knockout cells showed that, although the PKC pathway largely regulates FGF2-stimulated Runx2 activity by up-regulating Runx2 expression, it also modifies Runx2 protein post-translationally and thereby increases its transcriptional activity. Thus, we show for the first time that FGF/FGFR signaling stimulates the DNA-binding and transcriptional activities of Runx2 as well as its expression, and these are largely regulated by the PKC pathway.
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THE EFFECT OF FGF-MEDIATED FGFR SIGNALING ON THE EARLY MORPHOGENESIS AND MAINTENANCE OF THE CRANIAL SUTURE
THE JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY, 1999Co-Authors: Mihyun Park, Hyunmo RyooAbstract:Craniosynostosis, the premature fusion of cranial sutures, presumably involves disturbance of the interactions between different tissues within the cranial sutures. Interestingly, point mutaions in the genes encoding for the fibroblast growth factor receptors(FGFRs), especially FGFR2, cause various types of human craniosynostosis syndromes. To elucidate the function of these genes in the early morphogenesis of mouse cranial sutures, we first analyzed by in situ hybridization the expression of FGFR2(BEK) and osteopontin, an early marker of osteogenic differentiation, in the sagittal suture of calvaria during embryonic(E15-E18) and postnatal stage(P1-P3). FGFR2(BEK) was intensely expressed in the osteogenic fronts, whose cells undergo differentiation into osteoprogenitor cells that ultimately lay down the bone matrix. Osteopontin was expressed throughout the parietal bones excluding the osteogenic fronts, the periphery of the parietal bones. To further examine the role of FGF-mediated FGFR signaling in cranial suture, we did in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of FGF2 soaked beads onto both the osteogenic fronts and mid-mesenchyme of sagittal suture after 36 hours organ culture resulted in the increase of the tissue thickness and cell number around FGF2 beads, moreover FGF4-soaked beads implanted onto the osteogenic fronts stimulated suture closure due to an accelerated bone growth, compared to FGF4 beads placed onto mid-mesenchyme of sagittal suture and BSA control beads. In addition FGF2 induced the ectopic expression of osteopontin and Msx1 genes. Taken together, these data indicate that FGF-mediated FGFR signaling has a important role in regulating the cranial bone growth and maintenance of cranial suture, and suggest that FGF-mediated FGFR signaling is involved in regulating the balance between the cell proliferation and differentiation through inducing the expression of osteopontin and Msx1 genes.
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THE EFFECT OF FGF-MEDIATED FGFR SIGNALING ON THE EARLY MORPHOGENESIS AND MAINTENANCE OF THE CRANIAL SUTURE
THE JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY, 1999Co-Authors: Mihyun Park, Hyunmo RyooAbstract:Craniosynostosis, the premature fusion of cranial sutures, presumably involves disturbance of the interactions between different tissues within the cranial sutures. Interestingly, point mutaions in the genes encoding for the fibroblast growth factor receptors(FGFRs), especially FGFR2, cause various types of human craniosynostosis syndromes. To elucidate the function of these genes in the early morphogenesis of mouse cranial sutures, we first analyzed by in situ hybridization the expression of FGFR2(BEK) and osteopontin, an early marker of osteogenic differentiation, in the sagittal suture of calvaria during embryonic(E15-E18) and postnatal stage(P1-P3). FGFR2(BEK) was intensely expressed in the osteogenic fronts, whose cells undergo differentiation into osteoprogenitor cells that ultimately lay down the bone matrix. Osteopontin was expressed throughout the parietal bones excluding the osteogenic fronts, the periphery of the parietal bones. To further examine the role of FGF-mediated FGFR signaling in cranial suture, we did in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of FGF2 soaked beads onto both the osteogenic fronts and mid-mesenchyme of sagittal suture after 36 hours organ culture resulted in the increase of the tissue thickness and cell number around FGF2 beads, moreover FGF4-soaked beads implanted onto the osteogenic fronts stimulated suture closure due to an accelerated bone growth, compared to FGF4 beads placed onto mid-mesenchyme of sagittal suture and BSA control beads. In addition FGF2 induced the ectopic expression of osteopontin and Msx1 genes. Taken together, these data indicate that FGF-mediated FGFR signaling has a important role in regulating the cranial bone growth and maintenance of cranial suture, and suggest that FGF-mediated FGFR signaling is involved in regulating the balance between the cell proliferation and differentiation through inducing the expression of osteopontin and Msx1 genes.
David G. Fernig - One of the best experts on this subject based on the ideXlab platform.
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Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification
Open biology, 2016Co-Authors: Changye Sun, Mark C. Wilkinson, Edwin A. Yates, Chao Jiang, David G. FernigAbstract:The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans.
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HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors
PeerJ, 2015Co-Authors: Yong Li, Sarah E Taylor, Mark C. Wilkinson, David G. FernigAbstract:The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins.
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HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors
2014Co-Authors: Yong Li, Sarah E Taylor, Mark C. Wilkinson, David G. FernigAbstract:The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins.