Fibroblast Growth Factor 10-Fibroblast Growth Factor Receptor 2b Mediated Signaling Is Not Required for Adult Glandular Stomach Homeostasis

The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10''s main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.     

Loss of Dlg-1 in the Mouse Lens Impairs Fibroblast Growth Factor Receptor Signaling

Coordination of cell proliferation, differentiation and survival is essential for normal development and maintenance of tissues in the adult organism. Growth factor receptor tyrosine kinase signaling pathways and planar cell polarity pathways are two regulators of many developmental processes. We have previously shown through analysis of mice conditionally null in the lens for the planar cell polarity gene (PCP), Dlg-1, that Dlg-1 is required for fiber differentiation. Herein, we asked if Dlg-1 is a regulator of the Fibroblast growth factor receptor (Fgfr) signaling pathway, which is known to be required for fiber cell differentiation. Western blot analysis of whole fiber cell extracts from control and Dlg-1 deficient lenses showed that levels of the Fgfr signaling intermediates pErk, pAkt, and pFrs2α, the Fgfr target, Erm, and the fiber cell specific protein, Mip26, were reduced in the Dlg-1 deficient fiber cells. The levels of Fgfr2 were decreased in Dlg-1 deficient lenses compared to controls. Conversely, levels of Fgfr1 in Dlg-1 deficient lenses were increased compared to controls. The changes in Fgfr levels were found to be specifically in the triton insoluble, cytoskeletal associated fraction of Dlg-1 deficient lenses. Immunofluorescent staining of lenses from E13.5 embryos showed that expression levels of pErk were reduced in the transition zone, a region of the lens that exhibits PCP, in the Dlg-1 deficient lenses as compared to controls. In control lenses, immunofluorescent staining for Fgfr2 was observed in the epithelium, transition zone and fibers. By E13.5, the intensity of staining for Fgfr2 was reduced in these regions of the Dlg-1 deficient lenses. Thus, loss of Dlg-1 in the lens impairs Fgfr signaling and leads to altered levels of Fgfrs, suggesting that Dlg-1 is a modulator of Fgfr signaling pathway at the level of the receptors and that Dlg-1 regulates fiber cell differentiation through its role in PCP.     

Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity

Asparagine-linked glycosylation is an endoplasmic reticulum co- and post- translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.     

成纤维细胞生长因子与其受体相互作用及其抑制剂的研究进展

成纤维细胞生长因子（FGF）有许多重要的生理功能，并与肿瘤的形成有关.为了弄清FGF与成纤维细胞生长因子受体(FGFR)相互作用的机制，人们对FGF和FGFR的各个结合结构域进行了深入、细致的研究，定位了aFGF、bFGF的肝素结合区、bFGF的受体结合区、FGF受体的肝素结合区、配体结合区和FGF受体相互结合区，提出了两个FGF与FGFR相互作用的模型，在此基础上设计了FGF的核酸类、糖类和多肽类抑制剂，为寻找新一代抗癌药物打下了理论基础.     

Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor

Nectins belong to a family of immunoglobulin (Ig)-like cell-adhesion molecules comprising four members, nectin-1 through nectin-4. Nectins are involved in formation of the mechanical adhesive puncta adherentia junctions of synapses. Nectins share the same overall structural topology with an extracellular region containing three Ig modules, a transmembrane region, and a cytoplasmic region. In nectin-1, the first and second Ig module in the extracellular region are necessary for the trans-interaction with nectin-3 and formation of cis-dimers, respectively. The function of the third Ig module of nectin-1 remains unknown. We here report the structure in solution of the third, membrane-proximal Ig module of mouse nectin-1 (nectin-1 Ig3) solved by means of nuclear magnetic resonance (NMR) spectroscopy. It belongs to the C1 set of the Ig superfamily. Nectin-1 Ig3 was produced as a recombinant protein and induced neurite outgrowth in primary cultures of hippocampal and cerebellar granule neurons, an effect abolished by treatment with the fibroblast growth factor receptor (FGFR) inhibitor SU5402, or by transfection with a dominant-negative FGFR1 construct. We showed by surface plasmon resonance (SPR) analysis that nectin-1 Ig3 directly interacted with various isoforms of FGFR. Nectin-1 Ig3 induced phosphorylation of FGFR1c in the same manner as the whole nectin-1 ectodomain, and promoted survival of cerebellar granule neurons induced to undergo apoptosis. Finally, we constructed a peptide, nectide, by employing in silico modeling of various FGFR ligand-binding sites. Nectide mimicked all the effects of nectin-1 Ig3. We suggest that FGFR is a downstream signaling partner of nectin-1.     

Fibroblast Growth Factor Receptor 2c Signaling Is Required for Intestinal Cell Differentiation in Zebrafish

Background

There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.

Methodology/Principal Findings

We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mib^ta52b mutant that had defective Notch signaling.

Conclusions/Significance

In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mib^ta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.     

Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand–receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.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.     

Caenorhabditis elegans Fibroblast Growth Factor Receptor Signaling Can Occur Independently of the Multi-Substrate Adaptor FRS2

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y¹⁰⁰⁹ and Y¹⁰⁸⁷), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.FIBROBLAST growth factors (FGFs) play important roles in many developmental and physiological processes, including cell migration, angiogenesis, proliferation, differentiation, and survival (Ornitz and Itoh 2001; Polanska et al. 2009). Mammals have a battery of both FGF ligands and high-affinity receptors to carry out this diverse set of important functions. These ligands and their receptors are generated from a set of 18 genes encoding FGFs and 4 genes encoding their receptors (Eswarakumar et al. 2005). Upon ligand binding, fibroblast growth factor receptors (FGFRs) dimerize, activating their intrinsic tyrosine kinase activity, which causes both autophosphorylation on intracellular tyrosine residues and phosphorylation of additional substrates (Eswarakumar et al. 2005). These phosphorylation events lead to the assembly of a signaling complex around the activated receptor, ultimately promoting various downstream signaling pathways (Eswarakumar et al. 2005).A large portion of mammalian FGFR signaling is mediated by the multi-substrate adaptor protein FRS2/snt-1 (Kouhara et al. 1997; Hadari et al. 1998, 2001; Lax et al. 2002; Gotoh et al. 2005). FRS2 constitutively associates with the juxtamembrane region of the FGFR via its amino-terminal PTB domain (Xu et al. 1998; Ong et al. 2000). Upon FGFR activation, FRS2 becomes heavily phosphorylated, allowing it to recruit Grb2 and Shp2 via their SH2 domains (Kouhara et al. 1997; Eswarakumar et al. 2005). Since these components cannot associate with the receptor in the absence of FRS2 (Hadari et al. 2001), FRS2 serves as an essential link between the activated receptor and many downstream signal transduction pathways.The understanding of FGF-stimulated signal transduction pathways has been aided by the study of FGF signaling in model organisms. Powerful genetic screens and the reduced complexity of the set of FGFs and their receptors in both Drosophila melanogaster and Caenorhabditis elegans have helped promote an understanding of the conserved aspects of FGF signaling pathways (Huang and Stern 2005; Polanska et al. 2009). In C. elegans, FGF signaling is mediated by two FGF ligands, EGL-17 and LET-756, and a single FGF receptor, EGL-15 (DeVore et al. 1995; Burdine et al. 1997; Roubin et al. 1999). The EGL-15 FGFR is structurally very similar to mammalian FGF receptors, with the highest level of sequence conservation found within the intracellular tyrosine kinase domain and the three extracellular immunoglobulin (IG) domains.Similar to mammalian FGFRs, alternative splicing also generates functionally distinct EGL-15 isoforms. A major structural difference between EGL-15 and other FGFRs lies in an additional domain located between the first IG domain and the acid box of EGL-15. This EGL-15-specific insert is encoded by a pair of mutually exclusive fifth exons, generating two EGL-15 isoforms, 5A and 5B, with different functions (Goodman et al. 2003). Alternative splicing also affects the sequence at the very end of the carboxy-terminal domain (CTD) of EGL-15 (Goodman et al. 2003), giving rise to four distinct C-terminal isoform types, referred to as types I–IV (see Figure 1A).Open in a separate windowFigure 1.—The SEM-5∷EGL-15 interaction is dependent upon the SEM-5 SH2 binding sites in the CTD of EGL-15. (A) Important residues in the EGL-15 CTD isoforms. The C-terminal portion of the kinase domain and the CTD are shown. The gray box is common to all C-terminal isoforms. Sequences of the CTD isoforms can be found in Figure S5. (B) SEM-5 binds directly to EGL-15 Y1009 and Y1087. A full-length SEM-5 prey was mated to eight EGL-15 baits: empty vector control (pBTM116) and derivatives containing variants of either the type I or the type IV EGL-15(Intra). Growth is an indication of an interaction between SEM-5 and the EGL-15 bait. Dilutions of the culture mixture are indicated above. hFGFR1, a bait containing the corresponding portion of the intracellular domain of the human FGFR1. Similar results were obtained using the human SEM-5 ortholog, Grb2.EGL-15, like its mammalian orthologs, is also involved in a large variety of functions, including cell migration guidance affecting the sex myoblast (SM) (Stern and Horvitz 1991; DeVore et al. 1995; Goodman et al. 2003) and CAN cells (Fleming et al. 2005), muscle arm extension (Dixon et al. 2006), a number of processes controlling terminal axon morphology (Bulow et al. 2004), muscle protein degradation (Szewczyk and Jacobson 2003), and fluid homeostasis (Huang and Stern 2004). A conserved FGFR signaling pathway in C. elegans was established by identifying the genes necessary for the role of EGL-15 in fluid homeostasis, and much of this same pathway is utilized in other functions of EGL-15 (DeVore et al. 1995; Borland et al. 2001; Huang and Stern 2004, 2005). The identification of these genes was facilitated by a temperature-sensitive mutation affecting a receptor tyrosine phosphatase, CLR-1 (CLeaR), which functions to negatively regulate EGL-15 signaling (Kokel et al. 1998). Mutations in clr-1 abolish this regulatory constraint on EGL-15, resulting in fluid accumulation in the pseudocoelomic cavity due to hyperactive EGL-15 signaling. The buildup of this clear fluid, resulting in the Clr phenotype, is easily scored and can be used to identify suppressors (soc, suppressor of Clr) that reduce EGL-15 signaling efficiency. These suppressors define an EGL-15 signaling pathway necessary for fluid homeostasis, which involves the activation of the Ras/MAPK cascade via the SEM-5/Grb2 adaptor protein, the let-341/sos-1 Sos-like guanine nucleotide exchange factor, and a PTP-2-SOC-1/Shp2-Gab1 cassette (Borland et al. 2001).Mutations in egl-15 have been identified on the basis of their effects on either fluid homeostasis or the guidance of the migrating SMs (DeVore et al. 1995; Goodman et al. 2003). Most of these alleles fall into an allelic series and affect the general aspects of EGL-15 signaling (Goodman et al. 2003). The strongest of these alleles confers an early larval arrest phenotype, whereas weaker alleles confer either a scrawny body morphology (Scr) or just the ability to suppress the Clr phenotype (Soc). While the weakest of these alleles does not affect egg laying, more highly compromised mutants show an egg-laying defect due to the mispositioning of the SMs. Four egl-15 alleles specifically affect SM migration; when homozygous, these mutations cause dramatic mispositioning of the SMs, but do not cause a Soc phenotype (Goodman et al. 2003). Three of these are nonsense mutations in exon 5A and eliminate the 5A EGL-15 isoform. The phenotype of these mutants highlights the specific requirement of the 5A isoform for SM migration guidance. The fourth mutation in this class, egl-15(n1457), is a nonsense mutation that truncates the carboxy-terminal domain, specifically implicating the CTD in SM migration guidance.Immediately following their birth at the end of the first larval stage (L1), the two bilaterally symmetric SMs undergo anteriorly directed migrations to final positions that flank the precise center of the gonad (Sulston and Horvitz 1977). In the middle of the third larval stage, the SMs divide to generate 16 cells that differentiate into the egg-laying muscles. Multiple mechanisms help guide the migrations of the SMs (Chen and Stern 1998; Branda and Stern 2000), including a chemoattraction mediated by EGL-15 that guides the SMs to their precise final positions (Burdine et al. 1998). The EGL-17 FGF serves as the chemoattractive cue, emanating from central gonadal cells (Branda and Stern 2000). In the absence of this chemoattraction, SMs are posteriorly displaced (Stern and Horvitz 1991). While mispositioned SMs still generate sex muscles, these muscles end up too far posterior to attach properly, causing the animal to be defective in egg laying (Egl).The signal transduction pathway downstream of EGL-15 that mediates SM chemoattraction is not well established. Several lines of evidence implicate SEM-5/Grb2, LET-341/Sos, and LET-60/Ras in SM chemoattraction. The roles of components in the Ras-MAPK cascade in this event are less clear (Sundaram et al. 1996; Chen et al. 1997; Chen and Stern 1998). A crucial gap in our understanding lies in the link between activated EGL-15 and the downstream signaling components. Here we show that SEM-5, the C. elegans GRB2 ortholog, appears to bind directly to SH2 binding sites within the carboxy terminal tail of EGL-15. These interactions are required for SM chemoattraction, but not for the essential function of EGL-15.     

Cardiac-Specific Over-Expression of Epidermal Growth Factor Receptor 2 (ErbB2) Induces Pro-Survival Pathways and Hypertrophic Cardiomyopathy in Mice

Background

Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored.

Methodology/Principal Findings

We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2–3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib.

Conclusions/Significance

These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.     

A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.     

Novel Mechanisms of Fibroblast Growth Factor Receptor 1 Regulation by Extracellular Matrix Protein Anosmin-1

Activation of fibroblast growth factor (FGF) signaling is initiated by a multiprotein complex formation between FGF, FGF receptor (FGFR), and heparan sulfate proteoglycan on the cell membrane. Cross-talk with other factors could affect this complex assembly and modulate the biological response of cells to FGF. We have previously demonstrated that anosmin-1, a glycosylated extracellular matrix protein, interacts with the FGFR1 signaling complex and enhances its activity in an IIIc isoform-specific and HS-dependent manner. The molecular mechanism of anosmin-1 action on FGFR1 signaling, however, remains unknown. Here, we show that anosmin-1 directly binds to FGFR1 with high affinity. This interaction involves domains in the N terminus of anosmin-1 (cysteine-rich region, whey acidic protein-like domain and the first fibronectin type III domain) and the D2–D3 extracellular domains of FGFR1. In contrast, anosmin-1 binds to FGFR2IIIc with much lower affinity and displays negligible binding to FGFR3IIIc. We also show that FGFR1-bound anosmin-1, although capable of binding to FGF2 alone, cannot bind to a FGF2·heparin complex, thus preventing FGFR1·FGF2·heparin complex formation. By contrast, heparin-bound anosmin-1 binds to pre-formed FGF2·FGFR1 complex, generating an anosmin-1·FGFR1·FGF2·heparin complex. Furthermore, a functional interaction between anosmin-1 and the FGFR1 signaling complex is demonstrated by immunofluorescence co-localization and Transwell migration assays where anosmin-1 was shown to induce opposing effects during chemotaxis of human neuronal cells. Our study provides molecular and cellular evidence for a modulatory action of anosmin-1 on FGFR1 signaling, whereby binding of anosmin-1 to FGFR1 and heparin can play a dual role in assembly and activity of the ternary FGFR1·FGF2·heparin complex.FGF⁵ signaling plays an important role in a wide range of fundamental biological responses (1–3). Both FGF and FGFR bind to heparan sulfate (HS) and heparin, a highly sulfated type of HS produced in connective tissue mast cells. Heparan sulfate proteoglycans (HSPG) are the cell surface co-receptors essential for the formation of functional FGF·FGFR signaling complex (4, 5). There are four structurally related FGFRs (FGFR1–4), which consist of an extracellular ligand-binding region containing three immunoglobulin (Ig)-like domains (D1–D3), a single transmembrane domain, and a cytoplasmic domain with protein-tyrosine kinase catalytic activity. The 22 members of the FGF family bind to the interface formed by the D2/D3 domains and the linker between these domains (6, 7), whereas a conserved positively charged region in D2 serves as the HS binding site (8). An unusual stretch of seven to eight acidic residues designated as the “acid box” is present in the linker connecting D1 and D2. Alternative splicing events occur to generate various isoforms, including a truncated receptor lacking D1 and the D1–D2 linker or a full-length receptor that differs in the second half of D3, designated as IIIb and IIIc isoforms (5). Two crystal structures have been proposed to demonstrate how the FGF·FGFR·heparin complex is assembled (9, 10). Recent evidence suggests that both may be biologically relevant (11, 12).The diversity of FGF signaling pathways and consequent biological functions require that activation of FGFR should be tightly regulated. Such regulation can occur either at the level of the extracellular receptor-ligand complex assembly or via intracellular modulation of downstream effectors (13). Extracellular regulation mainly involves the interaction between each component of the FGF·FGFR·HS signaling complex. For example, FGF8 is shown to bind mostly to the FGFR IIIc isoforms, whereas FGF7 acts as the preferential ligand for the FGFR2 IIIb isoform (13, 14). Sequence specificity, length, and sulfation patterns of HS are also important regulators of the FGF·FGFR interaction (15, 16).Cell surface proteins other than FGFs and HSPGs participate in FGFR signaling regulation. FLRT3 (a member of the fibronectin-leucine-rich transmembrane protein family) promotes FGF signaling and interacts with FGFR1 and FGFR4 via its extracellular fibronectin type III (FnIII) domain (17). Sef (similar expression to fgf genes) functions as an antagonist of FGF signaling in zebrafish. The two FnIII regions of Sef are essential for its function and interaction with FGFR1 and FGFR2 (18). Neuronal cell adhesion molecule (NCAM), N-cadherin, and L1 have also been identified as functionally relevant in FGFR-mediated neurite outgrowth (19–22). The FnIII domains of NCAM bind to the D2 and D3 domains of FGFR1 (19) and FGFR2 (23) to induce ligand-independent receptor phosphorylation.Anosmin-1, an extracellular matrix-associated glycosylated protein, appears to be a novel member of the extracellular FGFR signaling modulators (24, 25). Loss-of-function mutations of anosmin-1 and FGFR1 are associated with Kallmann syndrome (KS), underlying X-linked, and autosomal dominant/recessive inheritance mode, respectively (26–28). KS is a human developmental genetic disorder characterized by loss of sense of smell (anosmia) caused by abnormal olfactory bulb development and delayed, even arrested puberty caused by disrupted migration of the gonadotropin-releasing hormone (GnRH)-secreting neuron. We previously reported that anosmin-1 acts as an FGFR1IIIc isoform-specific co-ligand, which enhances signaling activity. In human embryonic GnRH olfactory neuroblast FNC-B4 cells, anosmin-1 induced neurite outgrowth and cytoskeletal rearrangements through FGFR1-dependent mechanisms involving p42/44 and p38 mitogen-activated protein kinases and Cdc42/Rac1 activation (25). A functional interaction is also demonstrable between anosmin-1 and FGFR1 in optic nerve oligodendrocyte precursor development (24). Structurally, anosmin-1 comprises an N-terminal cysteine-rich domain (CR) and a whey acidic protein-like (WAP) domain, followed by four tandem FnIII repeats and a C-terminal histidine rich region (Fig. 1a). Current evidence suggests that anosmin-1 functions by affecting FGF2-induced activation of FGFR1 signaling rather than by directly stimulating the receptor. However, the precise molecular mechanism of this interaction remains unclear.Open in a separate windowFIGURE 1.Generation of recombinant anosmin-1, anosmin-1 mutants, FGFR1D1D3, and FGFR1D2D3 proteins. a, the schematic structures of recombinant proteins of anosmin-1 and FGFR1. Each domain in the wild type (PIWF4), point mutants (mPIWF4^N267K, mPIWF4^E514K, and mPIWF4^F517L), and truncated (PIWF1, PIWF2, and PIF4) anosmin-1 protein analogues are represented by a shaded rectangle. V5 and 6His epitopes at the C terminus are represented by a clear rectangle. Each immunoglobulin-like domain in the full ectodomain (FGFR1D1D3) and truncated form (FGFR1D2D3) of FGFR1 is represented by a half circle. The acid box (AB) is represented by a filled rectangle. H, histidine-rich region. b, 0.5–1 μg of purified recombinant proteins are loaded in each lane and visualized by colloidal blue staining. Molecular mass markers in kilodaltons are shown on the left.We now report for the first time that anosmin-1 directly binds to FGFR1 using surface plasmon resonance (SPR), chemical cross-linking, and immunofluorescence co-localization studies in living cells. This interaction occurs between the N-terminal CR, WAP, and the first FnIII domain of anosmin-1 and D2 and D3 ectodomains of FGFR1. Moreover, SPR studies using sequential injections and Transwell migration assays in immortalized FNC-B4-hTERT cells suggest that anosmin-1 can have opposing effects in the formation and activation of the FGF2·FGFR1·heparin complex depending on the order of their binding interactions with anosmin-1.     

A Mass Action Model of a Fibroblast Growth Factor Signaling Pathway and Its Simplification

We consider a kinetic law of mass action model for Fibroblast Growth Factor (FGF) signaling, focusing on the induction of the RAS-MAP kinase pathway via GRB2 binding. Our biologically simple model suffers a combinatorial explosion in the number of differential equations required to simulate the system. In addition to numerically solving the full model, we show that it can be accurately simplified. This requires combining matched asymptotics, the quasi-steady state hypothesis, and the fact subsets of the equations decouple asymptotically. Both the full and simplified models reproduce the qualitative dynamics observed experimentally and in previous stochastic models. The simplified model also elucidates both the qualitative features of GRB2 binding and the complex relationship between SHP2 levels, the rate SHP2 induces dephosphorylation and levels of bound GRB2. In addition to providing insight into the important and redundant features of FGF signaling, such work further highlights the usefulness of numerous simplification techniques in the study of mass action models of signal transduction, as also illustrated recently by Borisov and co-workers (Borisov et al. in Biophys. J. 89, 951–966, 2005, Biosystems 83, 152–166, 2006; Kiyatkin et al. in J. Biol. Chem. 281, 19925–19938, 2006). These developments will facilitate the construction of tractable models of FGF signaling, incorporating further biological realism, such as spatial effects or realistic binding stoichiometries, despite a more severe combinatorial explosion associated with the latter.     

Fibroblast Growth Factor Receptor 1 Amplification in Non-Small Cell Lung Cancer by Quantitative Real-Time PCR

Introduction

Amplification of the fibroblast growth factor receptor 1 (FGFR1) gene has been described in tumors of non-small-cell lung cancer (NSCLC) patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance.

Materials and Methods

We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application.

Results

In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025) for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption) as compared to light smokers.

Discussion

Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients’ survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.     

The Acidic Domain and First Immunoglobulin-Like Loop of Fibroblast Growth Factor Receptor 2 Modulate Downstream Signaling through Glycosaminoglycan Modification

Fibroblast growth factor receptors (FGFRs) are membrane-spanning tyrosine kinases that have been implicated in a variety of biological processes including mitogenesis, cell migration, development, and differentiation. We identified a unique isoform of FGFR2 expressed as a diffuse band with an unusually large molecular mass. This receptor is modified by glycosaminoglycan at a Ser residue located immediately N terminal to the acidic box, a stretch of acidic amino acids. The acidic box and the glycosaminoglycan modification site are encoded by an alternative exon of the FGFR2 gene. The acidic box appears to play an important role in glycosaminoglycan modification, and the presence of this domain is required for modification by heparan sulfate glycosaminoglycan. Moreover, the presence of the first immunoglobulin-like domain encoded by another alternative exon abrogated the modification. The high-affinity receptor with heparan sulfate modification enhanced receptor autophosphorylation, substrate phosphorylation, and ternary complex factor-independent gene expression. It also sustained mitogen-activated protein kinase activity and increased eventual DNA synthesis, a long-term response to fibroblast growth factor stimulation, at physiological ligand concentrations. We propose a novel regulation mechanism of FGFR2 signal transduction through glycosaminoglycan modification.     

Antagonism between Retinoic Acid and Fibroblast Growth Factor Signaling during Limb Development

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Multiple Signaling Pathways of the Insulin-Like Growth Factor 1 Receptor in Protection from Apoptosis

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.     

A New Avian Fibroblast Growth Factor Receptor in Myogenic and Chondrogenic Cell Differentiation

We studied the expression of FREK (fibroblast growth factor receptor-like embryonic kinase), a new receptor recently cloned from quail embryo, during the differentiation of skeletal muscle satellite cells and epiphyseal growth-plate chondrocytes. Although FREK mRNA was expressed in both cell types, satellite cells expressed higher levels of this mRNA than chondrocytes. FREK gene expression was found to be modulated by b-FGF in a biphasic manner: low concentrations increased expression, whereas high concentrations attenuated it. In both cell cultures, the levels of FREK mRNA declined during terminal differentiation. Moreover, retinoic acid (RA), which induces skeletal muscle satellite cells to differentiate, also caused a reduction in FREK gene expression in these cells. Induction of chondrocyte differentiation with ascorbic acid was monitored by a decrease in collagen type II gene expression and an increase in alkaline phosphatase activity. Satellite cell differentiation was marked by morphological changes as well as by increased sarcomeric myogenin content and creatine kinase activity and changes in the expression of the regulatory muscle-specific genes, MyoD and myogenin. DNA synthesis in both cell types was stimulated by b-FGF. However, in satellite cells, the response was bell-shaped, peaking at 1 ng/ml b-FGF, whereas in chondrocytes, higher levels of b-FGF were needed. b-FGF-dependent DNA synthesis in satellite cells was decreased by RA at concentrations over 10^-7M . The observed correlation between the level of FREK gene expression and various stages of differentiation, its modulation by b-FGF and RA, as well as the correlation between FREK gene expression and the physiological response to b-FGF, suggest that this specific FGF receptor plays an important role in muscle and cartilage cell differentiation.     

Structural Analysis of the Human Fibroblast Growth Factor Receptor 4 Kinase

The family of fibroblast growth factor receptors (FGFRs) plays an important and well-characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer, for example, breast cancer or rhabdomyosarcoma. Whereas FGFR1–FGFR3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study, we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type II inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4 inhibitors.     

Type 2 Fibroblast Growth Factor Receptor Signaling Preserves Stemness and Prevents Differentiation of Prostate Stem Cells from the Basal Compartment

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, which include basal, luminal, and neuroendocrine cells. Two types of P-SCs have been identified in both human and mouse adult prostates based on prostasphere or organoid cultures, cell lineage tracing, renal capsule implantation, and expression of luminal- and basal-specific proteins. The sphere-forming P-SCs are from the basal cell compartment that express P63, and are therefore designated as basal P-SCs (P-bSCs). Luminal P-SCs (P-lSCs) express luminal cytokeratins and Nkx3.1. Herein, we report that the type 2 FGF receptor (FGFR2) signaling axis is crucial for preserving stemness and preventing differentiation of P-bSCs. FGFR2 signaling mediated by FGFR substrate 2α (FRS2α) is indispensable for formation and maintenance of prostaspheres derived from P63⁺ P-bSCs. Ablation of Fgfr2 in P63⁺ cells in vitro causes the disintegration of prostaspheres. Ablation of Fgfr2 in vivo reduces the number of P63-expressing basal cells and enriches luminal cells. This suggests a basal stem cell-to-luminal cell differentiation. In addition, ablation of Fgfr2 in P63⁺ cells causes defective postnatal development of the prostate. Therefore, the data indicate that FGFR2 signaling is critical for preserving stemness and preventing differentiation of P-bSCs.