FGF signaling regulates development by processes beyond canonical pathways.

FGFs are key developmental regulators that engage a signal transduction cascade through receptor tyrosine kinases, prominently engaging ERK1/2 but also other pathways. However, it remains unknown whether all FGF activities depend on this canonical signal transduction cascade. To address this question, we generated allelic series of knock-in Fgfr1 and Fgfr2 mouse strains, carrying point mutations that disrupt binding of signaling effectors, and a kinase dead allele of Fgfr2 that broadly phenocopies the null mutant. When interrogated in cranial neural crest cells, we identified discrete functions for signaling pathways in specific craniofacial contexts, but point mutations, even when combined, failed to recapitulate the single or double null mutant phenotypes. Furthermore, the signaling mutations abrogated established FGF-induced signal transduction pathways, yet FGF functions such as cell-matrix and cell-cell adhesion remained unaffected, though these activities did require FGFR kinase activity. Our studies establish combinatorial roles of Fgfr1 and Fgfr2 in development and uncouple novel FGFR kinase-dependent cell adhesion properties from canonical intracellular signaling.

Distinct Requirements for FGFR1 and FGFR2 in Primitive Endoderm Development and Exit from Pluripotency.

Activation of the FGF signaling pathway during preimplantation development of the mouse embryo is known to be essential for differentiation of the inner cell mass and the formation of the primitive endoderm (PrE). We now show using fluorescent reporter knockin lines that Fgfr1 is expressed in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further show that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Finally, we document an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development.

Genetic insights into the mechanisms of Fgf signaling.

The fibroblast growth factor (Fgf) family of ligands and receptor tyrosine kinases is required throughout embryonic and postnatal development and also regulates multiple homeostatic functions in the adult. Aberrant Fgf signaling causes many congenital disorders and underlies multiple forms of cancer. Understanding the mechanisms that govern Fgf signaling is therefore important to appreciate many aspects of Fgf biology and disease. Here we review the mechanisms of Fgf signaling by focusing on genetic strategies that enable in vivo analysis. These studies support an important role for Erk1/2 as a mediator of Fgf signaling in many biological processes but have also provided strong evidence for additional signaling pathways in transmitting Fgf signaling in vivo.

Fgfr1 regulates development through the combinatorial use of signaling proteins.

Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo.

Receptor tyrosine kinases modulate distinct transcriptional programs by differential usage of intracellular pathways.

Receptor tyrosine kinases (RTKs) signal through shared intracellular pathways yet mediate distinct outcomes across many cell types. To investigate the mechanisms underlying RTK specificity in craniofacial development, we performed RNA-seq to delineate the transcriptional response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling in mouse embryonic palatal mesenchyme cells. While the early gene expression profile induced by both growth factors is qualitatively similar, the late response is divergent. Comparing the effect of MEK (Mitogen/Extracellular signal-regulated kinase) and PI3K (phosphoinositide-3-kinase) inhibition, we find the FGF response is MEK dependent, while the PDGF response is PI3K dependent. Furthermore, FGF promotes proliferation but PDGF favors differentiation. Finally, we demonstrate overlapping domains of PDGF-PI3K signaling and osteoblast differentiation in the palate and increased osteogenesis in FGF mutants, indicating this differentiation circuit is conserved in vivo. Our results identify distinct responses to PDGF and FGF and provide insight into the mechanisms encoding RTK specificity.

Characterization of rearrangements involving the ALK gene reveals a novel truncated form associated with tumor aggressiveness in neuroblastoma.

Activating mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma (NB), a cancer of the peripheral nervous system, and are thought to be the primary mechanism of oncogenic activation of this receptor in this pediatric neoplasm. To address the possibility that ALK activation may occur through genomic rearrangements as detected in other cancers, we first took advantage of high-resolution array-comparative genomic hybridization to search for ALK rearrangements in NB samples. Using complementary experiments by capture/paired-end sequencing and FISH experiments, various types of rearrangements were fully characterized, including partial gains or amplifications, in several NB cell lines and primary tumors. In the CLB-Bar cell line, we described a genomic rearrangement associated with an amplification of the ALK locus, leading to the expression of a 170 kDa protein lacking part of the extracellular domain encoded by exons 4 to 11, named ALK(Δ4-11). Analysis of genomic DNA from the tumor at diagnosis and relapse revealed that the ALK gene was amplified at diagnosis but that the rearranged ALK allele was observed at the relapse stage only, suggesting that it may be implicated in tumor aggressiveness. Consistently, oncogenic and tumorigenic properties of the ALK(Δ4-11) variant were shown after stable expression in NIH3T3 cells. Moreover, we documented an increased constitutive kinase activity of this variant, as well as an impaired maturation and retention into intracellular compartments. These results indicate that genomic rearrangements constitute an alternative mechanism to ALK point mutations resulting in receptor activation.

Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment.

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

The PKR activator PACT is induced by Aß: involvement in Alzheimer's disease.

The neuropathological hallmarks of Alzheimer's disease (AD) include senile plaques made of Aß peptide, neurofibrillary tangles containing hyperphosphorylated tau protein and neuronal loss. The pro-apoptotic kinase PKR can be activated by Aß and can phosphorylate tau protein via GSK3ß kinase activation. The activated form of PKR (pPKR) accumulates in affected neurons and could participate in neuronal degeneration in AD. The mechanism of abnormal PKR activation in AD is not elucidated but could be linked to the PKR activator PACT. PACT stainings, and levels were assessed in the brains of AD patients and in APP/PS1 knock-in transgenic mice and in cell cultures exposed to stresses. We showed that PACT and pPKR colocalizations are enhanced in AD brains. Their levels are increased and correlated in AD and APP/PS1 knock-in mice brains. In human neuroblastoma cells exposed to Aß, tunicamycin or H2O2, PACT and pPKR concentrations are increased. PACT then PKR inhibitions indicate that PACT is upstream of PKR activation. Our findings demonstrate that PACT levels are enhanced in AD brains and could partly be caused by the action of Aß. In addition, PACT participates in PKR activation. The PACT-PKR pathway represents a potential link between Aß accumulation, PKR activation and tau phosphorylation.

Modulation of tau phosphorylation by the kinase PKR: implications in Alzheimer's disease.

Double-stranded RNA dependent kinase (PKR) is a pro-apoptotic kinase that controls protein translation. Previous studies revealed that activated PKR is increased in brains with Alzheimer's disease (AD). Glycogen Synthase Kinase Aß (GSK-3ß) is responsible for tau phosphorylation and controls several cellular functions also including apoptosis. The goal of this work was to determine if PKR could concurrently trigger GSK-3ß activation, tau phosphorylation and apoptosis. In AD brains, both activated kinases co-localize with phosphorylated tau in neurons. In SH-SY5Y cell cultures, tunicamycin and Aß(1-42) activate PKR, GSK-3ß and induce tau phosphorylation and all these processes are attenuated by PKR inhibitors or PKR siRNA. Our results demonstrate that neuronal PKR co-localizes with GSK-3ß and tau in AD brains and is able to modulate GSK-3ß activation, tau phosphorylation and apoptosis in neuroblastoma cells exposed to tunicamycin or Aß. PKR could represent a crucial signaling point relaying stress signals to neuronal pathways leading to cellular degeneration in AD.

Activation of the orphan receptor tyrosine kinase ALK by zinc.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates.

In contrast to agonist monoclonal antibodies, both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in neuroblastoma and glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor in vertebrates.