Fibroblast Growth Factor Signaling Research Articles

Prevention of vascular dementia via immunotherapeutic blockade of renin-angiotensin system in a rat model

IntroductionAs several clinical trials have revealed that angiotensin-converting enzyme inhibitors and angiotensin II (Ang II) receptor blockers may be efficient in treating vascular dementia (VaD), the long-acting blockade of the renin-angiotensin system (RAS) would be useful considering the poor adherence of antihypertensive drugs. Accordingly, we continuously blocked RAS via vaccination and examined the effectiveness of the VaD model in rats. MethodsMale Wistar rats were exposed to two-vessel occlusions (2VO) after three injections of Ang II peptide vaccine. The effects of the vaccine were evaluated in the novel object recognition test, brain RAS components, and markers for oligodendrocytes. ResultsIn the vaccinated rats, anti-Ang II antibody titer level was increased in serum until Day 168, but not in cerebral parenchyma. Vaccinated rats showed better object recognition memory with inhibited demyelination in the corpus callosum and activation of astrocytes and microglia. Also, levels of BrdU/GSTπ-positive cells and the phosphorylation of cAMP response element binding protein was increased in vaccinated rats, indicating that the differentiation of oligodendrocyte progenitor cells to mature oligodendrocytes was accelerated. Vaccinated rats showed increased expression of fibroblast growth factor-2 (FGF2), which was observed in endothelial cells. Angiotensinogen mRNA was decreased at 7 days after 2VO but increased at 14 and 28 days. ConclusionAng II vaccine might have promoted oligodendrocyte differentiation and inhibited astrocytic and microglial activation by stimulating FGF2 signaling in the endothelial cells-oligodendrocyte/astrocyte/microglia coupling. These data indicate the feasibility of Ang II vaccine for preventing progression of vascular dementia.

Responses of Epibranchial Placodes to Disruptions of the FGF and BMP Signaling Pathways in Embryonic Mice.

Placodes are ectodermal thickenings of the embryonic vertebrate head. Their descendants contribute to sensory organ development, but also give rise to sensory neurons of the cranial nerves. In mammals, the signaling pathways which regulate the morphogenesis and neurogenesis of epibranchial placodes, localized dorsocaudally to the pharyngeal clefts, are poorly understood. Therefore, we performed mouse whole embryo culture experiments to assess the impact of pan-fibroblast growth factor receptor (FGFR) inhibitors, anti-FGFR3 neutralizing antibodies or the pan-bone morphogenetic protein receptor (BMPR) inhibitor LDN193189 on epibranchial development. We demonstrate that each of the three paired epibranchial placodes is regulated by a unique combination of FGF and/or bone morphogenetic protein (BMP) signaling. Thus, neurogenesis depends on fibroblast growth factor (FGF) signals, albeit to different degrees, in all epibranchial placodes (EP), whereas only EP1 and EP3 significantly rely on neurogenic BMP signals. Furthermore, individual epibranchial placodes vary in the extent to which FGF and/or BMP signals (1) have access to certain receptor subtypes, (2) affect the production of Neurogenin (Ngn)2+ and/or Ngn1+ neuroblasts, and (3) regulate either neurogenesis alone or together with structural maintenance. In EP2 and EP3, all FGF-dependent production of Ngn2+ neuroblasts is mediated via FGFR3 whereas, in EP1, it depends on FGFR1 and FGFR3. Differently, production of FGF-dependent Ngn1+ neuroblasts almost completely depends on FGFR3 in EP1 and EP2, but not in EP3. Finally, FGF signals turned out to be responsible for the maintenance of both placodal thickening and neurogenesis in all epibranchial placodes, whereas administration of the pan-BMPR inhibitor, apart from its negative neurogenic effects in EP1 and EP3, causes only decreases in the thickness of EP3. Experimentally applied inhibitors most probably not only blocked receptors in the epibranchial placodes, but also endodermal receptors in the pharyngeal pouches, which act as epibranchial signaling centers. While high doses of pan-FGFR inhibitors impaired the development of all pharyngeal pouches, high doses of the pan-BMPR inhibitor negatively affected only the pharyngeal pouches 3 and 4. In combination with partly concordant, partly divergent findings in other vertebrate classes our observations open up new approaches for research into the complex regulation of neurogenic placode development.

FGF-2–dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2-dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

365P The analysis of FGFR-gene family alterations in glioma

Fibroblast growth factor (FGFR) alterations are implicated across a range of solid tumors, promoting oncogenesis as a result of amplification, mutations, and structural variations. The FGFR gene family consists of four highly conserved transmembrane tyrosine kinase receptors (FGFR1–4). FGFR signaling influences angiogenesis and tumor cell migration, differentiation, proliferation, and survival. Recently, the cIMPACT-NOW released update 4 and update research, which considered FGFR alterations as a marker in brain tumor classification and prognosis.

Functional Roles of FGF Signaling in Early Development of Vertebrate Embryos.

Fibroblast growth factors (FGFs) comprise a large family of growth factors, regulating diverse biological processes including cell proliferation, migration, and differentiation. Each FGF binds to a set of FGF receptors to initiate certain intracellular signaling molecules. Accumulated evidence suggests that in early development and adult state of vertebrates, FGFs also play exclusive and context dependent roles. Although FGFs have been the focus of research for therapeutic approaches in cancer, cardiovascular disease, and metabolic syndrome, in this review, we mainly focused on their role in germ layer specification and axis patterning during early vertebrate embryogenesis. We discussed the functional roles of FGFs and their interacting partners as part of the gene regulatory network for germ layer specification, dorsal–ventral (DV), and anterior-posterior (AP) patterning. Finally, we briefly reviewed the regulatory molecules and pharmacological agents discovered that may allow modulation of FGF signaling in research.

Canopy Fibroblast Growth Factor Signaling Regulator 2 (CNPY2) Inhibits Neuron Apoptosis in Parkinson's Disease via the AKT/GSK3β Pathway.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic neurons. Canopy fibroblast growth factor signaling regulator 2 (CNPY2) is down-regulated in this disease, but its functions are unknown. This study investigates the effects and regulation of CNPY2 in the apoptosis of neurons in PD. We established a PD model in vivo by a five consecutive days-injection of 1-methyl-4- phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to mice. In vitro, the human SH-SY5Y neuroblastoma cells, after differentiation, were treated with 1-Methyl-4-phenylpyridinium iodide (MPP+) for modeling. The cells were transfected with a recombinant vector overexpressing CNPY2 followed by MPP+ treatment. Expression of CNPY2 and proteins related to apoptosis was detected by real-time PCR, western blot, or immunofluorescence staining. The ROS level and mitochondrial membrane potential were determined by flow cytometry. Cell viability and apoptosis were measured by MTT assay and TUNEL staining. CNPY2 level was down-regulated both in the brain and retina of PD mice and also inhibited in neurons by MPP+ in vitro. Overexpression of CNPY2 repressed the level of Bax and cleaved caspase-3, enhanced Bcl-2 level, and promoted neurite length under MPP+ treatment. CNPY2 overexpression reduced the accumulation of ROS and mitochondria dysfunction in neurons. The AKT/ GSK3β signaling pathway was activated by overexpressed CNPY2 to inhibit MPP+-induced neuronal apoptosis, which was confirmed using an AKT inhibitor MK-2206 2HCl. CNPY2 alleviates oxidative stress, mitochondria dysfunction, and apoptosis of neurons induced by MPP+ by activating the AKT/ GSK3β signaling pathway.

A Review on Molecular Factors Affecting Stomach Development

Stomach as a highly important portion of the GIT which is responsible for storage, digestion of food, immune defense, and hormonal organization of metabolic homeostasis; it originates from posterior foregut. The transcription factors such as Forkhead Box A (Foxa1/2/3), Gata Binding Protein (Gata4/6), Hnf1 Homeobox B (Hnf1b), and Sry- Box Transcription Factor 2 (Sox2) have a crucial role in gastric specification. Endodermal foregut patterning and regionalization, mesenchymal differentiation and cell lineage specification of stomach are affected by many essential factors such as Retinoic acid (RA) signalling, Wingless-related integration site (Wnt) signals, fibroblast growth factor (FGF) signals, Bone morphogenetic protein (BMP) signalling, sonic hedgehog (Shh) signalling and GATA family .There is a lack for understanding the organ lineage relationships and lineage-specific markers during stomach development and differentiation. The objectives of this review are to focus on the normal development and the mesenchymal differentiation of the stomach and to highlight the relatively limited information we have about genes and transcription factors associated with stomach specification, regionalization, and differentiation of component cell lineages. Keywords: Stomach, Specification, Regionalization, Transcription Factor, Lineage-specific markers, Genes, Cell lineage specification. Read more →

Separating the BRCA1 and BRCA2 phenotype: a genomic pathway analysis

<h3>Objectives:</h3> Emerging data suggests that key differences exist between <i>BRCA1</i> and <i>BRCA2</i> associated ovarian cancer, including response to therapy and survival outcomes. The purpose of this study was to identify gene expression profiles and interacting pathways unique to <i>BRCA1-</i> and <i>BRCA2-</i> associated high grade serous ovarian cancer (HGSOC) samples compared to one another as well as to <i>BRCA</i> wild type, homologous recombination proficient (HRP) tumors. <h3>Methods:</h3> Of 657 total HGSOC samples, 15 <i>BRCA2</i> mutated (2.2%), 16 (2.4%) <i>BRCA1</i> mutated, and 375 (57%) HRP samples were analyzed. <i>BRCA</i> mutated was defined as somatic variants that result in loss of function. The HRP control group was defined as samples negative for aberrations in <i>BRCA1/2</i> and 28 HR genes. Gene expression data was collected from Tempus and unpaired t-tests were used to identify differentially expressed genes (DEG) with p-value <0.05 and fold change (FC) of 1.5. Meta and pathway analyses were performed among <i>BRCA1</i>, <i>BRCA2</i> and HRP groups using Venn diagram and Advaita Bio's iPathwayGuide. <h3>Results:</h3> From 18,284 genes with measured expression, 843 (4.6%) DEG were found between <i>BRCA2</i> vs <i>BRCA1,</i> 748 (4.1%) between <i>BRCA2</i> vs HRP and 1,858 (10.2%) between <i>BRCA1</i> and HRP. 8,296 mutated genes were similarly expressed in both <i>BRCA2</i> and <i>BRCA1</i> groups with FC < 1.5, including DNA damage response genes <i>ATM</i> (log2FC = -0.43, p=0.51), <i>CHEK2</i> (log2FC= -0.21, p=0.79), <i>TP53</i> (log2FC = -0.73, p=0.52) and <i>PARP1</i> (log2FC=0.62, p=0.26). For <i>BRCA1</i> compared to HRP group, 214 miRNAs and 137 gene upstream regulators were identified unique to <i>BRCA1</i>; top identified pathways were metabolism related. For <i>BRCA2</i> compared to HRP group, no miRNAs and 108 gene upstream regulators were identified; identified pathways were Wnt signaling related. On meta-analysis of the 3 comparisons, 550 DEGs were found for <i>BRCA2</i> and 82 DEG for <i>BRCA1</i> that were not shared by HRP group (Figure 1). Pathway analysis revealed significant involvement of Wnt signaling pathway unique to <i>BRCA2</i> group compared to fibroblast growth factor signaling and PI3K-Akt signaling for <i>BRCA1</i>. <h3>Conclusions:</h3> Our study identified genomic signatures for <i>BRCA2</i> versus <i>BRCA1</i> associated ovarian cancer, not shared by control HRP tumors in a sample representative of HGSOC tumor heterogeneity. Results suggest <i>BRCA1/2</i> should not be considered a single entity, but rather separate phenotypes each with unique opportunities for targeted therapy.

Human Ipsc-Based Signaling Disruption Reporter Assay for Teratogenic Toxicity of Chemicals

The number of man-made chemicals has increased exponentially in recent decades, and some of them induce foetal malformations. Because complex and precisely programmed signaling pathways play important roles in developmental processes, their disruption by external chemicals often triggers developmental toxicity. However, highly accurate and high-throughput screening assays for the teratogenic potential of various chemicals are currently lacking. In this study, we propose a reporter assay that utilises human induced pluripotent stem cells (iPSCs) to detect changes in fibroblast growth factor signaling, which is essential for limb morphogenesis. The dynamics of this signaling after an exposure to a chemical were integrated to estimate the degree of signaling disruption, which afforded a good prediction of the capacity of chemicals listed in the ECVAM International Validation Study to induce limb malformation. Our results indicate that the human iPSC-based signaling disruption assay could be useful for the initial screening of potentially teratogenic chemicals.

Fibroblast growth factorand kidney disease: Updates for emerging novel therapeutics.

The discovery of fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) provided a profound new insight into physiological and metabolic functions. FGF has a large family by having divergent structural elements and enable functional divergence and specification. FGF and FGFRs are highly expressed during kidney development. Signals from the ureteric budregulate morphogenesis, nephrogenesis, and nephron progenitor survival. Thus, FGF signaling plays an important rolein kidney progenitor cell aggregation at the sites of new nephron formation. This review will summarize the current knowledge about functions of FGF signaling in kidney development and their ability to promote regeneration in injured kidneys and its use as a biomarker and therapeutic target in kidney diseases. Further studies are essential to determine the predictive significance of the various FGF/FGFR deviations and to integrate them into clinical algorithms.

Abstract 1012: In vitro antitumor effect of AZD4547 in ovarian cancer cell lines

Abstract Of all the gynecological cancer, ovarian cancer is the most fatal malignancy. In particular, strong chemotherapy resistance in ovarian cancer raises the need for new therapeutic strategy. Since fibroblast growth factor (FGF) signaling pays an important role in malignant transformation, tumor growth, angiogenesis, and chemoresistance, fibroblast growth factor receptor (FGFR) inhibitors are being assessed in clinical trials for several FGFR-driven cancer. FGFRs is known to be highly dysregulated in ovarian cancer, however the effect of FGFR inhibition in ovarian cancer has not been elucidated. In this study, we investigated the anti-tumor effects of AZD4547, an FGFR1-4 inhibitor, in various types of ovarian cancer cell line. AZD4547 markedly inhibited proliferation and stimulated apoptosis of ovarian cancer cells. In addition, AZD4547 inhibited cell migration and invasion under non-toxic condition. Moreover, AZD4547 reduced the sphere forming ability and the self-renewal capacities of ovarian cancer stem cells (CSCs) which are strongly resistant to paclitaxel. Furthermore, AZD4547 exhibited anti-angiogenic effect on human umbilical vein endothelial cells. Based on these anti-tumor effect of AZD4547 in ovarian cancer cell lines, we suggests AZD4547 as a promising agent for ovarian cancer treatment. Citation Format: Ji Hae Seo, Seungmee Lee, Chi-Heum Cho, So-Jin Shin. In vitro antitumor effect of AZD4547 in ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1012.

Abstract 1458: Targeting fibroblast growth factor receptors in castration-resistant prostate cancer

Abstract Background: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. Although AR signaling inhibitors (ARSI), such as enzalutamide (ENZ) and abiraterone (ABI), extend survival in recurrent and castration-resistant prostate cancer (CRPC), durable complete responses are rare. Bypass mechanisms employed by metastatic CRPC include amplification of AR and AR splice variants in AR-high CRPC (ARPC) and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We showed previously that DNPC bypasses AR-dependence through fibroblast growth factor (FGF) signaling. However, the utility of targeting the FGF pathway in other molecular subtypes of CRPC remains to be determined. Methods: RNASeq was conducted on 98 patient metastases, 18 LuCaP patient-derived xenograft (PDX) models and CRPC cell lines. Sensitivity to ARSI and FGFR inhibitors (FGFRi) alone or in combination in cell lines (C4-2B, VCaP, 22Rv1, DU145 and NCIH660) or ex vivo PDX tumor cells (10 ARPC models, 2 DNPC models, and 6 SCNPC models) was measured through CellTitre Glo assays. On-target effects of inhibitors were assessed through immunoblot and qPCR. In vivo efficacy of FGFRi in ARPC and SCNPC was conducted using LuCaP PDX models. Results: Interrogation of FGF pathway activity and FGFR expression using RNASeq and FGF signature scores in CRPC metastases, LuCaP PDX models and CRPC cell lines revealed upregulated FGF pathway activation in AR-null DNPC and SCNPC and in subsets of ARPC. In vitro/ex vivo analysis of FGFRi (erdafitinib, CH5183284 and rogaratinib) demonstrated robust growth suppression in DNPC and moderate growth suppression in SCNPC. In vivo CH5183284 administration recapitulated the growth suppression observed in vitro for SCNPC LuCaP 93 and LuCaP 173.1 but not for SCNPC LuCaP 49. Furthermore, in vitro/ex vivo analysis of AR-expressing CRPC models determined that combination ARSI (ENZ, ABI or darolutamide) with FGFRi was superior to ARSI or FGFRi monotherapies in a subset of models. Immunoblot and qPCR analyses verified that FGFRi-mediated growth suppression occurs through inactivation of the MAPK/MEK and ERK signal transduction cascade in AR-expressing CRPC. Finally, ARPC LuCaP PDX models treated with combination ARSI and FGFRi showed significant tumor growth inhibition compared to control and monotherapy groups in vivo. Analysis of resistance mechanisms to FGFRi therapy are ongoing. Conclusions: The emergence of bypass mechanisms that subvert AR-dependence in mCRPC highlights a need for novel therapeutic strategies. Our data nominate the FGF pathway as an actionable target in diverse phenotypes of treatment-refractory mCRPC. Citation Format: Mark P. Labrecque, Lisha G. Brown, Ilsa M. Coleman, Bryce Lakely, Holly M. Nguyen, Eva Corey, Peter S. Nelson, Colm Morrissey. Targeting fibroblast growth factor receptors in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1458.

High Expression of FGF2/SDC1 Signaling Contributes to Poor Clinical Outcome in Hodgkin Lymphoma Patients and SDC1 siRNA Suppresses Proliferation, Migration, and Invasion in Hodgkin Lymphoma Cells

Our recent studies have shown that Fibroblast Growth Factor-2 (FGF2) and Syndecan-1 (SDC1) expressions are upregulated, by 45- and 24-fold respectively, in CD30+ cells from tissues of relapsed versus relapsed-free Hodgkin lymphoma (HL) patients (p<0.005; Fig. 1A). Normal control and relapsed-free groups showed similar FGF2 and SDC1 expression profiles in tissue samples. The circulating HL cells in peripheral blood lymphocytes (PBL) have demonstrated that FGF2 and SDC1 mRNA were upregulated, by nearly 7000- and 9000-fold respectively, in relapsed versus relapsed-free HL patient samples (p<0.0001; Fig. 1B). Preliminary Kaplan-Meier analyses indicate that the HL patient group (n=4) with elevated co-expression of FGF2 and SDC1 had shortened survival by six-fold when compared to the majority of patients (n=11) with significantly lowered expression of FGF2/SDC1 (p < 0.05). This suggests that high levels of FGF2/SDC1 contribute to poor prognosis in HL patients. Western blot analyses showed that shed SDC1 regulate the HL cell division rate by a negative feedback mechanism. However, secretion of FGF2 and an abundance of four FGF receptor (FGFR) proteins were not associated with the cell division rate.Among eleven HL cell lines examined, HD-MyZ represented the best model for relapsed/refractory HL due to its highest cell proliferation rate (35 hour doubling time) and overexpression of FGF2 and SDC1, by 60- and 150-fold respectively, when compared with the B lymphoblast cell line, C1RB7. Immunofluorescence and co-immunoprecipitation studies in normal fibroblasts and HL cells demonstrate that FGF2 and SDC1 are co-localized on the cell membrane and bind together as ligand and receptor. Additionally, FGF2 binds to FGFRs in HL cells with lower affinity than in normal fibroblasts with predominant binding to FGFR1, suggesting that FGF2 signaling largely involves both SDC1 and FGFR1. Fluorescence microscopy studies showed that FGFR1 is constitutively phosphorylated in HL cells, and it stimulates cell proliferation by an autocrine pathway. Treatment with exogenous FGF2 in the absence of serum resulted in phosphorylation of both AKT and ERK, thereby activating FGF2-mediated cell proliferation. FGF2 induction also upregulated mRNA expressions of SDC1 (12-fold), proliferation marker- CCND1 (242-fold) and metastatic marker- MMP9 (67-fold) compared to unstimulated cells.To examine the molecular mechanism, whereby SDC1 is involved in the proliferation and metastasis of HL cells, SDC1 knockdown (KD) was achieved near 100% with 1nM siRNA after 48 hours of transfection. The SDC1 KD condition did not affect cell morphology but significantly attenuated cell proliferation by two-fold (p < 0.05; Fig. 2A) by downregulating BCL2 (87%), CCND1 (74%), and CDK4 (64%), indicating that SDC1 has pivotal functional roles in proliferation of HL cells. Transwell chamber assays showed that SDC1 KD reduced migration of HL cells by two-fold (p < 0.05; Fig. 2B) and invasion by three-fold (p < 0.005; Fig. 2C) by downregulating metastatic markers MMP9, ADAM9, ITGB3, and VEGF . The flow cytometry data demonstrated that 50% of HL cells expressed residual SDC1 on the cell membrane after SDC1 KD for 48 hours, which potentially maintains the normal HL cells morphology and cellular functions. However, four days of prolonged SDC1 KD caused an induction of apoptosis by upregulating CASP3, and continuous KD for 17 days caused near eradication of surviving HL cells.In conclusion, our results provide evidence that increased FGF2/SDC1 expression and signaling may drive and maintain the hyperproliferation of HL cells, thereby contributing to disease progression and poor prognosis in HL patients. Therefore, a novel method to inhibit altered FGF2/SDC1 signaling offers an excellent potential for treating high-risk and refractory HL patient populations. [Display omitted] DisclosuresGoy:Genentech: Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics / J&J: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Feldman:Janssen: Speakers Bureau; Celgene: Speakers Bureau; AbbVie: Speakers Bureau; Bristol-Myers Squibb: Consultancy; Kite Pharma: Speakers Bureau; Seattle Genetics: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Speakers Bureau. Leslie:KITE pharma: Speakers Bureau; celgene: Speakers Bureau; seattle genetics: Speakers Bureau. Pecora:COTA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Caladrius Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Zebrafish Minichromosome Maintenance Protein 5 Gene Regulates the Development and Migration of Facial Motor Neurons via Fibroblast Growth Factor Signaling

Minichromosome maintenance protein 5 (MCM5), a member of the microchromosomal maintenance protein family, plays an important role in the initiation and extension of DNA replication. However, its role in neural development in zebrafish remains unclear. Here, we used morpholino (MO) and CRISPR/Cas9 to knock down mcm5 and investigated the developmental features of facial motor neurons (FMNs) in the hindbrain of zebrafish. We found that knockdown of mcm5 using mcm5 MO resulted in a small head, small eyes, and a blurred midbrain-hindbrain boundary, while MO injection of mcm5 led to decrease in FMNs and their migration disorder. However, the mutant of mcm5 only resulted in the migration defect of FMNs rather than quantity change. We further investigated the underlying mechanism of mcm5 in the development of hindbrain using in situ hybridization (ISH) and fgfr1a mRNA co-injected with mcm5 MO. Results from ISH showed that the fibroblast growth factor (FGF) signaling pathway was changed when the MCM5 function was lost, with the decrease in fgfr1a and the increase in fgf8, while that of pea3 had opposite trend. FMN development defects were rescued by fgfr1a mRNA co-injected with mcm5 MO. Our results demonstrated that FGF signaling pathway is required for FMN development in zebrafish. Specifically, mcm5 regulates FMN development during zebrafish growing.

Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine‐tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling‐dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR‐mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF‐mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.

Effects of FGFR Tyrosine Kinase Inhibition in OLN-93 Oligodendrocytes.

Fibroblast growth factor (FGF) signaling is involved in the pathogenesis of multiple sclerosis (MS). Data from neuropathology studies suggest that FGF signaling contributes to the failure of remyelination in MS. In MOG35–55-induced EAE, oligodendrocyte-specific deletion of FGFR1 and FGFR2 resulted in a less severe disease course, reduced inflammation, myelin and axon degeneration and changed FGF/FGFR and BDNF/TrkB signaling. Since signaling cascades in oligodendrocytes could not be investigated in the EAE studies, we here aimed to characterize FGFR-dependent oligodendrocyte-specific signaling in vitro. FGFR inhibition was achieved by application of the multi-kinase-inhibitor dovitinib and the FGFR1/2/3-inhibitor AZD4547. Both substances are potent inhibitors of FGF signaling; they are effective in experimental tumor models and patients with malignancies. Effects of FGFR inhibition in oligodendrocytes were studied by immunofluorescence microscopy, protein and gene analyses. Application of the tyrosine kinase inhibitors reduced FGFR1, phosphorylated ERK and Akt expression, and it enhanced BDNF and TrkB expression. Furthermore, the myelin proteins CNPase and PLP were upregulated by FGFR inhibition. In summary, inhibition of FGFR signaling in oligodendrocytes can be achieved by application of tyrosine kinase inhibitors. Decreased phosphorylation of ERK and Akt is associated with an upregulation of BDNF/TrkB signaling, which may be responsible for the increased production of myelin proteins. Furthermore, these data suggest that application of FGFR inhibitors may have the potential to promote remyelination in the CNS.

Structural and Functional Characterization of the FGF Signaling Pathway in Regeneration of the Polychaete Worm Alitta virens (Annelida, Errantia).

Epimorphic regeneration of lost body segments is a widespread phenomenon across annelids. However, the molecular inducers of the cell sources for this reparative morphogenesis have not been identified. In this study, we focused on the role of fibroblast growth factor (FGF) signaling in the posterior regeneration of Alitta virens. For the first time, we showed an early activation of FGF ligands and receptor expression in an annelid regenerating after amputation. The expression patterns indicate that the entire regenerative bud is competent to FGFs, whose activity precedes the initiation of cell proliferation. The critical requirement of FGF signaling, especially at early stages, is also supported by inhibitor treatments followed by proliferation assay, demonstrating that induction of blastemal cells depends on FGFs. Our results show that FGF signaling pathway is a key player in regenerative response, while the FGF-positive wound epithelium, ventral nerve cord and some mesodermal cells around the gut could be the inducing tissues. This mechanism resembles reparative regeneration of vertebrate appendages suggesting such a response to the injury may be ancestral for all bilaterians.

Kindlin2 regulates neural crest specification via integrin-independent regulation of the FGF signaling pathway.

The focal adhesion protein Kindlin2 is essential for integrin activation, a process that is fundamental to cell-extracellular matrix adhesion. Kindlin 2 (Fermt2) is widely expressed in mouse embryos, and its absence causes lethality at the peri-implantation stage due to the failure to trigger integrin activation. The function of kindlin2 during embryogenesis has not yet been fully elucidated as a result of this early embryonic lethality. Here, we showed that kindlin2 is essential for neural crest (NC) formation in Xenopus embryos. Loss-of-function assays performed with kindlin2-specific morpholino antisense oligos (MOs) or with CRISPR/Cas9 techniques in Xenopus embryos severely inhibit the specification of the NC. Moreover, integrin-binding-deficient mutants of Kindlin2 rescued the phenotype caused by loss of kindlin2, suggesting that the function of kindlin2 during NC specification is independent of integrins. Mechanistically, we found that Kindlin2 regulates the fibroblast growth factor (FGF) pathway, and promotes the stability of FGF receptor 1. Our study reveals a novel function of Kindlin2 in regulating the FGF signaling pathway and provides mechanistic insights into the function of Kindlin2 during NC specification.

The Multifunctional Contribution of FGF Signaling to Cardiac Development, Homeostasis, Disease and Repair.

The current focus on cardiovascular research reflects society’s concerns regarding the alarming incidence of cardiac-related diseases and mortality in the industrialized world and, notably, an urgent need to combat them by more efficient therapies. To pursue these therapeutic approaches, a comprehensive understanding of the mechanism of action for multifunctional fibroblast growth factor (FGF) signaling in the biology of the heart is a matter of high importance. The roles of FGFs in heart development range from outflow tract formation to the proliferation of cardiomyocytes and the formation of heart chambers. In the context of cardiac regeneration, FGFs 1, 2, 9, 16, 19, and 21 mediate adaptive responses including restoration of cardiac contracting rate after myocardial infarction and reduction of myocardial infarct size. However, cardiac complications in human diseases are correlated with pathogenic effects of FGF ligands and/or FGF signaling impairment. FGFs 2 and 23 are involved in maladaptive responses such as cardiac hypertrophic, fibrotic responses and heart failure. Among FGFs with known causative (FGFs 2, 21, and 23) or protective (FGFs 2, 15/19, 16, and 21) roles in cardiac diseases, FGFs 15/19, 21, and 23 display diagnostic potential. The effective role of FGFs on the induction of progenitor stem cells to cardiac cells during development has been employed to boost the limited capacity of postnatal cardiac repair. To renew or replenish damaged cardiomyocytes, FGFs 1, 2, 10, and 16 were tested in (induced-) pluripotent stem cell-based approaches and for stimulation of cell cycle re-entry in adult cardiomyocytes. This review will shed light on the wide range of beneficiary and detrimental actions mediated by FGF ligands and their receptors in the heart, which may open new therapeutic avenues for ameliorating cardiac complications.

Impact of aging on gene expression response to x-ray irradiation using mouse blood

As a radiation biodosimetry tool, gene expression profiling is being developed using mouse and human peripheral blood models. The impact of dose, dose-rate, and radiation quality has been studied with the goal of predicting radiological tissue injury. In this study, we determined the impact of aging on the gene expression profile of blood from mice exposed to radiation. Young (2 mo) and old (21 mo) male mice were irradiated with 4 Gy x-rays, total RNA was isolated from whole blood 24 h later, and subjected to whole genome microarray analysis. Pathway analysis of differentially expressed genes revealed young mice responded to x-ray exposure by significantly upregulating pathways involved in apoptosis and phagocytosis, a process that eliminates apoptotic cells and preserves tissue homeostasis. In contrast, the functional annotation of senescence was overrepresented among differentially expressed genes from irradiated old mice without enrichment of phagocytosis pathways. Pathways associated with hematologic malignancies were enriched in irradiated old mice compared with irradiated young mice. The fibroblast growth factor signaling pathway was underrepresented in older mice under basal conditions. Similarly, brain-related functions were underrepresented in unirradiated old mice. Thus, age-dependent gene expression differences should be considered when developing gene signatures for use in radiation biodosimetry.