Extracellular point mutations in FGFR2 result in elevated ERK1/2 activation and perturbation of neuronal differentiation

Two independent gain-of-function point mutations (S252W and P253R) in the extracellular region of the FGFR2 (fibroblast growth factor receptor 2) increase the binding affinity for the growth factor. The effect of this enhanced growth factor binding by these mutants is expected to be an increase in activation of regular signalling pathways from FGFR2 as a result of more receptors being engaged by ligand at any given time. Using PC12 (pheochromocytoma) cells as a model cell system we investigated the effect of these mutations on protein phosphorylation including the receptor, the activation of downstream signalling pathways and cell differentiation. Our results show that the effects of both of these extracellular mutations have unexpected intracellular phenotypes and cellular responses. Receptor phosphorylation was altered in both the ligand-stimulated and unstimulated states. The mutants also resulted in differential phosphorylation of a number of intracellular proteins. Both mutations resulted in enhanced ERK1/2 (extracellular-signalregulated kinase1/2) activation. Although ERK1/2 activation is believed to transduce signals resulting in cell differentiation, this response was abrogated in the cells expressing the mutant receptors. The results of the present study demonstrate that single extracellular point mutations in the FGFR2 have a profound effect on intracellular signalling and ultimately on cell fate.     

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT₂ receptor (5-HT₂R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT₂R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Gα_q/11 and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Gα_q/11, 5-HT₂R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT₂R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT₂R-mediated FGFR2 transactivation in C6 cells.     

Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.     

Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation

Mutations in fibroblast growth factor receptors (Fgfrs) 1-3 cause skeletal disease syndromes in humans. Although these Fgfrs are expressed at various stages of chondrocyte and osteoblast development, their function in specific skeletal cell types is poorly understood. Using conditional inactivation of Fgfr1 in osteo-chondrocyte progenitor cells and in differentiated osteoblasts, we provide evidence that FGFR1 signaling is important for different stages of osteoblast maturation. Examination of osteogenic markers showed that inactivation of FGFR1 in osteo-chondro-progenitor cells delayed osteoblast differentiation, but that inactivation of FGFR1 in differentiated osteoblasts accelerated differentiation. In vitro osteoblast cultures recapitulated the in vivo effect of FGFR1 on stage-specific osteoblast maturation. In immature osteoblasts, FGFR1 deficiency increased proliferation and delayed differentiation and matrix mineralization, whereas in differentiated osteoblasts, FGFR1 deficiency enhanced mineralization. Furthermore, FGFR1 deficiency in differentiated osteoblasts resulted in increased expression of Fgfr3, a molecule that regulates the activity of differentiated osteoblasts. Mice lacking Fgfr1, either in progenitor cells or in differentiated osteoblasts, showed increased bone mass as adults. These data demonstrate that signaling through FGFR1 in osteoblasts is necessary to maintain the balance between bone formation and remodeling through a direct effect on osteoblast maturation.     

The dual roles of geminin during trophoblast proliferation and differentiation

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.     

Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.     

Cannabinoid 1 receptor-dependent transactivation of fibroblast growth factor receptor 1 emanates from lipid rafts and amplifies extracellular signal-regulated kinase 1/2 activation in embryonic cortical neurons

G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.     

Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases

Inhibition of the functions of L1 cell adhesion molecule (L1) by ethanol has been implicated in the pathogenesis of the neurodevelopmental aspects of the fetal alcohol syndrome (FAS). Ethanol at pharmacological concentrations has been shown to inhibit L1-mediated neurite outgrowth of rat post-natal day 6 cerebellar granule cells (CGN). Extracellular signal-related kinases (ERK) 1/2 activation occurs following L1 clustering. Reduction in phosphoERK1/2 by inhibition of mitogen-activated protein kinase kinase (MEK) reduces neurite outgrowth of cerebellar neurons. Here, we examine the effects of ethanol on L1 activation of ERK1/2, and whether this activation occurs via activation of fibroblast growth factor receptor 1 (FGFR1). Ethanol at 25 mm markedly inhibited ERK1/2 activation by both clustering L1 with cross-linked monoclonal antibodies, or by L1-Fc chimeric proteins. Clustering L1 with subsequent ERK1/2 activation did not result in tyrosine phosphorylation of the FGFR1. In addition, inhibition of FGFR1 tyrosine kinase blocked basic fibroblast growth factor (bFGF) activation of ERK1/2, but did not affect activation of ERK1/2 by clustered L1. We conclude that ethanol disrupts the signaling pathway between L1 clustering and ERK1/2 activation, and that this occurs independently of the FGFR1 pathway in cerebellar granule cells.     

Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault

Fibroblast growth factor receptors (FGFRs) play major roles in skeletogenesis, and activating mutations of the human FGFR1, FGFR2 and FGFR3 genes cause premature fusion of the skull bones (craniosynostosis). We have investigated the patterns of expression of Fgfr1, Fgfr2 and Fgfr3 in the fetal mouse head, with specific reference to their relationship to cell proliferation and differentiation in the frontal and parietal bones and in the coronal suture. Fgfr2 is expressed only in proliferating osteoprogenitor cells; the onset of differentiation is preceded by down-regulation of Fgfr2 and up-regulation of Fgfr1. Following up-regulation of the differentiation marker osteopontin, Fgfr1, osteonectin and alkaline phosphatase are down-regulated, suggesting that they are involved in the osteogenic differentiation process but not in maintaining the differentiated state. Fgfr3 is expressed in the cranial cartilage, including a plate of cartilage underlying the coronal suture, as well as in osteogenic cells, suggesting a dual role in skull development. Subcutaneous insertion of FGF2-soaked beads onto the coronal suture on E15 resulted in up-regulation of osteopontin and Fgfr1 in the sutural mesenchyme, down-regulation of Fgfr2, and inhibition of cell proliferation. This pattern was observed at 6 and 24 hours after bead insertion, corresponding to the timing and duration of FGF2 diffusion from the beads. We suggest (a) that a gradient of FGF ligand, from high levels in the differentiated region to low levels in the environment of the osteogenic stem cells, modulates differential expression of Fgfr1 and Fgfr2, and (b) that signalling through FGFR2 regulates stem cell proliferation whereas signalling through FGFR1 regulates osteogenic differentiation.     

Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCγ signaling pathway

Basic fibroblast growth factor (basic FGF) has pivotal roles in the function of various cell types. Here, we report the effects of basic FGF in the regulation of dental pulp stem cell (DPSC) behaviors including maintaining stemness and directing differentiation. Cells isolated from human dental pulp tissues exhibited stem cell properties including the expression of mRNA markers for embryonic and mesenchymal stem cells, the expression of Stro-1, and the multipotential differentiation. Basic FGF stimulated colony-forming units of DPSCs and up-regulated the expression of the embryonic stem cell markers; Oct4, Rex-1, and Nanog. Moreover, osteogenic medium containing basic FGF inhibited alkaline phosphatase enzymatic activity and mineralization of DPSCs. On the contrary, basic FGF appeared to be an influential growth factor in the neurogenic differentiation of DPSCs. In the presence of basic FGF, increased DPSCs neurosphere size and the up-regulation of neurogenic markers were noted. Inhibitors of FGFR or PLCγ were able to ablate the basic FGF-induced neuronal differentiation of DPSCs. Taken together, these results suggest basic FGF may be involved in the mechanisms controlling DPSCs cell fate decisions.     

Differences in signal transduction pathways by which platelet-derived and fibroblast growth factors activate extracellular signal-regulated kinase in differentiating oligodendrocytes

Treatment of cultured rat oligodendroglial progenitors with either platelet-derived growth factor (PDGF) or fibroblast growth factor-2 (FGF-2) activated extracellular signal regulated kinase 2 (ERK2). Activation was transient in response to PDGF, whereas it was greater and more prolonged in response to FGF-2. ERK2 activation by PDGF was preceded by a very rapid, robust and transient tyrosine phosphorylation of the PDGF receptor. Although there was consistently more activation of ERK2 in response to FGF-2 than to PDGF, immunostaining of FGF receptors 1 (FGFR1) and 2 (FGFR2) and their tyrosine phosphorylation in progenitors was very weak, and both receptors were up-regulated during differentiation to oligodendrocytes. Tyrosine phosphorylation of the FGF receptors was maximal from 15 to 60 min of treatment and was sustained for many hours. Binding of radioiodinated FGF-2 to FGFR1 was predominant in progenitors, whereas binding to FGFR2 was predominant in oligodendrocytes. ERK2 activation by PDGF was more sensitive to inhibition of tyrosine kinases, whereas ERK2 activation by FGF-2 was relatively more sensitive to inhibitors of protein kinase C. These differences in signal transduction pathways probably contribute to the different cellular responses of oligodendroglial lineage cells to PDGF and FGF-2, respectively.     

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation

Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.     

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. In conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.