FGFs/FGFRs系统对骨调节的研究进展

成纤维细胞生长因子家族(fibroblast growth factors,FGFs)及其受体FGFRs系统影响骨骼发育和形成过程,FGF与细胞表面FGFR结合,激活信号通路调控多种细胞生长、分化和凋亡。骨是FGF的重要靶器官,研究表明FGFs/FGFRs系统对骨组织成骨细胞、破骨细胞、软骨细胞的增殖和分化起重要调控作用,本文就FGFs/FGFRs系统对骨组织调节研究进展进行综述。     

Small molecule inhibition of fibroblast growth factor receptors in cancer

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs), which are a sub-family of the superfamily of receptor tyrosine kinases, to regulate human development and metabolism. Uncontrolled FGF signaling is responsible for diverse array of developmental disorders, most notably skeletal syndromes due to FGFR gain-of-function mutations. Studies in the last few years have provided significant evidence for the importance of FGF signaling in the pathogenesis of diverse cancers, including endometrial and bladder cancers. FGFs are both potent mitogenic and angiogenic factors and can contribute to carcinogenesis by stimulating cell proliferation and tumor angiogenesis. Gene knockout and pharmacological inhibition of FGFRs in in vivo and in vitro models validate FGFRs as a target for cancer treatment. Considerable efforts are being expended to develop specific, small-molecule inhibitors for treating FGFR-driven cancers. Recent reviews on the FGF/FGFR system have focused primarily on signaling, pathophysiology, and functions in cancer. In this article, we review the key roles of FGFR in cancer, provide an update on the status of clinical trials with small-molecule FGFR inhibitors, and discuss how the current structural data on FGFR kinases guide the design and characterization of new FGFR inhibitors.     

Structural basis for fibroblast growth factor receptor activation

FGF signaling plays a ubiquitous role in human biology as a regulator of embryonic development, homeostasis and regenerative processes. In addition, aberrant FGF signaling leads to diverse human pathologies including skeletal, olfactory, and metabolic disorders as well as cancer. FGFs execute their pleiotropic biological actions by binding, dimerizing and activating cell surface FGF receptors (FGFRs). Proper regulation of FGF-FGFR binding specificity is essential for the regulation of FGF signaling and is achieved through primary sequence variations among the 18 FGFs and seven FGFRs. The severity of human skeletal syndromes arising from mutations that violate FGF-FGFR specificity is a testament to the importance of maintaining precision in FGF-FGFR specificity. The discovery that heparin/heparan sulfate (HS) proteoglycans are required for FGF signaling led to numerous models for FGFR dimerization and heralded one of the most controversial issues in FGF signaling. Recent crystallographic analyses have led to two fundamentally different models for FGFR dimerization. These models differ in both the stoichiometry and minimal length of heparin required for dimerization, the quaternary arrangement of FGF, FGFR and heparin in the dimer, and in the mechanism of 1:1 FGF-FGFR recognition and specificity. In this review, we provide an overview of recent structural and biochemical studies used to differentiate between the two crystallographic models. Interestingly, the structural and biophysical analyses of naturally occurring pathogenic FGFR mutations have provided the most compelling and unbiased evidences for the correct mechanisms for FGF-FGFR dimerization and binding specificity. The structural analyses of different FGF-FGFR complexes have also shed light on the intricate mechanisms determining FGF-FGFR binding specificity and promiscuity and also provide a plausible explanation for the molecular basis of a large number craniosynostosis mutations.     

The pleiotropic effects of fibroblast growth factor receptors in mammalian development

In recent years the study of fibroblast growth factor receptors (FGFRs) in normal development and human genetic disorders has increased our understanding of some complex cellular processes. At least fifteen genetic disorders result from mutations within FGFR genes including skeletal dysplasias such as Apert syndrome and achondroplasia. In vitro experiments and the generation of animal models indicate that these mutations result in activation of the receptors and that FGFRs act as negative regulators of bone growth. FGFRs also play a role in wound healing and cancer. In this article, we review the expression of FGFRs in human development, the phenotypes resulting from FGFR mutations, and recent data identifying pathways downstream of the activated receptors.     

Aberrant fibroblast growth factor receptor signaling in bladder and other cancers

Fibroblast growth factors (FGFs) are potent mitogens, morphogens, and inducers of angiogenesis, and FGF signaling governs the genesis of diverse tissues and organs from the earliest stages. With such fundamental embryonic and homeostatic roles, it follows that aberrant FGF signaling underlies a variety of diseases. Pathological modifications to FGF expression are known to cause salivary gland aplasia and autosomal dominant hypophosphatemic rickets, while mutations in FGF receptors (FGFRs) result in a range of skeletal dysplasias. Anomalous FGF signaling is also associated with cancer development and progression. Examples include the overexpression of FGF2 and FGF6 in prostate cancer, and FGF8 overexpression in breast and prostate cancers. Alterations in FGF signaling regulators also impact tumorigenesis, which is exemplified by the down-regulation of Sprouty 1, a negative regulator of FGF signaling, in prostate cancer. In addition, several FGFRs are mutated in human cancers (including FGFR2 in gastric cancer and FGFR3 in bladder cancer). We recently identified intriguing alterations in the FGF pathway in a novel model of bladder carcinoma that consists of a parental cell line (TSU-Pr1/T24) and two sublines with increasing metastatic potential (TSU-Pr1-B1 and TSU-Pr1-B2), which were derived successively through in vivo cycling. It was found that the increasingly metastatic sublines (TSU-Pr1-B1 and TSU-Pr1-B2) had undergone a mesenchymal to epithelial transition. FGFR2IIIc expression, which is normally expressed in mesenchymal cells, was increased in the epithelial-like TSU-Pr1-B1 and TSU-Pr1-B2 sublines and FGFR2 knock-down was associated with the reversion of cells from an epithelial to a mesenchymal phenotype. These observations suggest that modified FGF pathway signaling should be considered when studying other cancer types.     

Fibroblast growth factors and their receptors in the central nervous system

Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues. Among the 23 FGF members, ten have been identified in the brain. Four FGF receptors (FGFRs), receptor tyrosine kinases, are known so far. Ligand binding of these receptors greatly depends on the presence of heparan sulfate proteoglycans, which act as low affinity FGFRs. Ligand binding specificity of FGFRs depends on the third extracellular Ig-like domain, which is subject to alternative splicing. Activation of FGFRs triggers several intracellular signaling cascades. These include phosphorylation of src and PLC leading finally to activation of PKC, as well as activation of Crk and Shc. SNT/FRS2 serves as an alternative link of FGFRs to the activation of PKC and, in addition, activates the Ras signaling cascade. In the CNS, FGFs are widely expressed; FGF-2 is predominantly synthesized by astrocytes, whereas other FGF family members, e.g., FGF-5, FGF-8, and FGF-9, are primarily synthesized by neurons. During CNS development FGFs play important roles in neurogenesis, axon growth, and differentiation. In addition, FGFs are major determinants of neuronal survival both during development and during adulthood. Adult neurogenesis depends greatly on FGF-2. Finally, FGF-1 and FGF-2 seem to be involved in the regulation of synaptic plasticity and processes attributed to learning and memory.     

FGF and FGFR signaling in chondrodysplasias and craniosynostosis

The first experimental mouse model for FGF2 in bone dysplasia was made serendipitously by overexpression of FGF from a constitutive promoter. The results were not widely accepted, rightfully drew skepticism, and were difficult to publish; because of over 2,000 studies published on FGF‐2 at the time (1993), only a few reported a role of FGF‐2 in bone growth and differentiation. However, mapping of human dwarfisms to mutations of the FGFRs shortly, thereafter, made the case that bone growth and remodeling was a major physiological function for FGF. Subsequent production of numerous transgenic and targeted null mice for several genes in the bone growth and remodeling pathways have marvelously elucidated the role of FGFs and their interactions with other genes. Indeed, studies of the FGF pathway present one of the best success stories for use of experimental genetics in functionally parsing morphogenetic regulatory pathways. What remains largely unresolved is the pleiotropic nature of FGF‐2. How does it accelerate growth in one cell then stimulate apoptosis or retard growth for another cell in the same type of tissue? Some of the answers may come through distinguishing the FGF‐2 protein isoforms, made from alternative translation start sites, these appear to have substantially different functions. Although we have made substantial progress, there is still much to be learned regarding FGF‐2 as a most complex, enigmatic protein. Studies of genetic models in mice and human FGFR mutations have provided strong evidence that FGFRs are important modulators of osteoblast function during membranous bone formation. However, there is some controversy regarding the effects of FGFR signaling in human and murine genetic models. Although significant progress has been made in our understanding of FGFR signaling, several questions remain concerning the signaling pathways involved in osteoblast regulation by activated FGFR. Additionally, little is known about the specific role of FGFR target genes involved in cranial bone formation. These issues need to be addressed in future in in vitro and in vivo approaches to better understand the molecular mechanisms of action of FGFR signaling in osteoblasts that result in anabolic effects in bone formation. J. Cell. Biochem. © 2005 Wiley‐Liss, Inc.     

Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning.

Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid-gestational expression of a novel secreted kinase-deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant-negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane-tethered kinase-deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant-negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild-type mouse embryos may help regulate FGF activity during normal development.     

FGF8在脊椎动物早期胚胎发育中的调控作用

成纤维细胞生长因子8 (fibroblast growth factor 8,FGF8)是成纤维细胞生长因子家族的成员之一,是一种组织发育过程中的重要分泌性调控信号分子,参与脊椎动物的多种组织器官的发生与发育.早期胚胎细胞通过表达FGF8在组织和器官发育、血管发生、血细胞生成、附肢发生和伤口愈合等方面发挥着重要作用.FGF8不但可以在细胞外通过胞内信号通路,而且也可以进入细胞内部发挥生物学功能.本文就FGF8在脊椎动物神经系统、内脏器官、肢体发育及不对称发育等组织、器官发育中的调控作用予以阐述.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

FGFs in endochondral skeletal development

The mammalian skeleton developments and grows through two complementary pathways: membranous ossification, which gives rise to the calvarial bones and distal clavicle, and endochondral ossification, which is responsible for the bones of the limbs, girdles, vertebrae, face and base of the skull and the medial clavicle. Fibroblast growth factors (FGFs) and their cognate FGF receptors (FGFRs) play important roles in regulating both pathways. However, the details of how FGF signals are initiated, propagated and modulated within the developing skeleton are only slowly emerging. This prospect will focus on the current understanding of these events during endochondral skeletal development with special attention given to concepts that have emerged in the past few years. J. Cell. Biochem. 110: 1046–1057, 2010. Published 2010 Wiley‐Liss, Inc.     

FGF signaling in the developing endochondral skeleton

Mutations in fibroblast growth factor receptors (Fgfrs) are the etiology of many craniosynostosis and chondrodysplasia syndromes in humans. The phenotypes associated with these human syndromes and the phenotypes resulting from targeted mutagenesis in the mouse have defined essential roles for FGF signaling in both endochondral and intramembranous bone development. In this review, I will focus on the role of FGF signaling in chondrocytes and osteoblasts and how FGFs regulate the growth and development of endochondral bone.     

Intracellular retention, degradation, and signaling of glycosylation-deficient FGFR2 and craniosynostosis syndrome-associated FGFR2C278F

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are known to play a critical role in a variety of fundamental processes, including wound healing, angiogenesis, and development of multiple organ systems. Mutations in the FGFR gene family have been linked to a series of syndromes (the craniosynostosis syndromes) whose primary phenotype involves aberrant development of the craniofacial skeleton. Craniosynostosis syndrome-linked FGFR mutations have been shown to be gain of function in terms of receptor activation and have been presumed to result in increased levels of FGF/FGFR signaling. Unfortunately, studies attempting to link expression of mutant FGFRs with changes in cellular phenotype have yielded conflicting results. In an effort to better understand the biochemical consequences of these mutations on receptor function, here we have investigated the effect of the FGFR2C278F mutation of Crouzon craniosynostosis syndrome on receptor trafficking, ubiquitination, degradation, and signaling. We find that FGFR2C278F exhibits diminished glycosylation, increased degradation, and limited cellular sublocalization in the osteoblastic cell line, MC3T3E1(C4). Additionally, we show that trafficking and autoactivation of wild type FGFR2 is glycosylation-dependent. Both FGFR2C278F and unglycosylated wild type FGFR2 signal through phospholipase Cgamma in a ligand-independent manner as well as exhibit dramatically increased binding to the adaptor protein, Frs2. These findings suggest that autoactive FGFR2 can signal from intracellular compartments. Based upon our results, we propose that the functional signaling of craniosynostosis mutant, autoactive receptors is limited in some cell types by protective cellular responses, such as increased trafficking to lysosomes and proteasomes for degradation.     

Evolutionarily conserved and divergent expression of members of the FGF receptor family among vertebrate embryos, as revealed by FGFR expression patterns in Xenopus

Fibroblast growth factors (FGFs) mediate many cell-cell signaling events during early development. While the actions of FGFs have been well-studied, the roles played by specific members of the FGF receptor (FGFR) family are poorly understood. To characterize the roles played by individual FGFRs we compared the regulation and expression of the three Xenopus FGFRs described to date (XFGFR-1, XFGFR-2, and XFGFR-4). First, we describe the expression of Xenopus FGFR-4; XFGFR-4 is present as a maternal mRNA and is found in the embryo through at least the tadpole stage. XFGFR-4 and XFGFR-1 mRNAs are present at comparable levels, arguing that both mediate FGF signaling during early development. Second, the expression of XFGFR-4 in animal caps differs from the expression of XFGFR-1 and XFGFR-2, suggesting that the FGFRs are independently regulated in ectoderm. Third, using whole-mount in situ hybridization, we show that XFGFR-1, XFGFR-2, and XFGFR-4 are expressed in dramatically different patterns, arguing that specific FGF signaling events are mediated by different members of the FGFR family. Among these, FGF signaling during the induction of neural crest cells is likely to be mediated by XFGFR-4. Comparison of our results with previously reported FGFR expression patterns reveals that FGFR-1 expression is highly conserved among vertebrate embryos, and FGFR-2 expression shows many features that are conserved and some that are divergent. In contrast, the expression pattern of FGFR-4 is highly divergent among vertebrate embryos. Received: 5 August 1999 / Accepted: 18 January 2000     

Fibroblast growth factors and their receptors in cancer

FGFs (fibroblast growth factors) and their receptors (FGFRs) play essential roles in tightly regulating cell proliferation, survival, migration and differentiation during development and adult life. Deregulation of FGFR signalling, on the other hand, has been associated with many developmental syndromes, and with human cancer. In cancer, FGFRs have been found to become overactivated by several mechanisms, including gene amplification, chromosomal translocation and mutations. FGFR alterations are detected in a variety of human cancers, such as breast, bladder, prostate, endometrial and lung cancers, as well as haematological malignancies. Accumulating evidence indicates that FGFs and FGFRs may act in an oncogenic fashion to promote multiple steps of cancer progression by inducing mitogenic and survival signals, as well as promoting epithelial-mesenchymal transition, invasion and tumour angiogenesis. Therapeutic strategies targeting FGFs and FGFRs in human cancer are therefore currently being explored. In the present review we will give an overview of FGF signalling, the main FGFR alterations found in human cancer to date, how they may contribute to specific cancer types and strategies for therapeutic intervention.     

Fibroblast growth factor signaling controlling bone formation: An update

Fibroblast Growth Factors (FGFs) regulate prenatal and postnatal bone formation through activation of FGF receptors (FGFR) and downstream signaling events. During the last decade, major advances have been made in our understanding of the mechanisms by which FGF/FGFR signaling controls osteoprogenitor cell replication and osteoblast differentiation and function. The analysis of the phenotype induced by FGF invalidation and mutations in FGFR allowed to delineate key FGF signaling pathways that regulate osteoblastogenesis. Molecular genomic studies led to identify target genes that are controlled by FGF/FGFR signaling and govern osteoblasts. The analysis of intracellular signaling pathways showed the importance of functional crosstalks between FGF signaling and other pathways in the regulation of bone formation. These recent progresses in the mechanisms underlying FGF/FGFR signaling may provide a molecular basis for developing therapeutic strategies in human skeletal dysplasias.     

Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.     

The roles of the FGF signal in zebrafish embryos analyzed using constitutive activation and dominant-negative suppression of different FGF receptors

The roles of the FGF family growth factors and their receptors (FGFRs) in zebrafish embryos were examined using variously modified versions of the four FGFR genes (fgfr1–4). Constitutively active forms of all of the examined FGFRs (ca-FGFRs) caused dorsalization, brain caudalization, and secondary axis formation, indicating that the main FGF signal transduction downstream of the receptor is highly similar among FGFRs. All of the membrane-bound type of dominant-negative FGFRs (mdn-FGFRs) derived from the four fgfr genes, which interfere with endogenous FGFRs, produced posterior truncation, as previously reported in both Xenopus and zebrafish. mdn-FGFR3c had the strongest effects on embryos, progressively disrupting the posterior structure as the dose increased. At the highest dose, only the forebrain was formed. At lower doses, mdn-FGFR3c mainly suppressed the paraxial mesoderm. The co-injection of mRNA for different mdn-FGFRs and FGFs resulted in diverse suppression spectra of the respective FGFRs against FGFs. Only mdn-FGFR3c severely suppressed all of the FGFs examined. We also examined the effects of the secretory type of dominant-negative FGFRs (sdn-FGFRs), which are released from cells and trap FGF ligands. Only sdn-FGFR3c resulted in the characteristic effect of selectively disrupting the isthmic development, as well as the tailbud. The co-injection of the mRNA for sdn-FGFRs and FGFs suggested that sdn-FGFR3c inhibits FGFs of the FGF8 subfamily, which is consistent with its specific effects on development. We discuss the implications of our findings obtained in the present study.