FGF4 and FGF9 have synergistic effects on odontoblast differentiation.

The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

FGF4 protects the liver from nonalcoholic fatty liver disease by activating the AMP-activated protein kinase-Caspase 6 signal axis.

BACKGROUND AND AIMS: NAFLD represents an increasing health problem in association with obesity and diabetes with no effective pharmacotherapies. Growing evidence suggests that several FGFs play important roles in diverse aspects of liver pathophysiology. Here, we report a previously unappreciated role of FGF4 in the liver. APPROACH AND RESULTS: Expression of hepatic FGF4 is inversely associated with NAFLD pathological grades in both human patients and mouse models. Loss of hepatic Fgf4 aggravates hepatic steatosis and liver damage resulted from an obesogenic high-fat diet. By contrast, pharmacological administration of recombinant FGF4 mitigates hepatic steatosis, inflammation, liver damage, and fibrogenic markers in mouse livers induced to develop NAFLD and NASH under dietary challenges. Such beneficial effects of FGF4 are mediated predominantly by activating hepatic FGF receptor (FGFR) 4, which activates a downstream Ca2+ -Ca2+ /calmodulin-dependent protein kinase kinase beta-dependent AMP-activated protein kinase (AMPK)-Caspase 6 signal axis, leading to enhanced fatty acid oxidation, reduced hepatocellular apoptosis, and mitigation of liver damage. CONCLUSIONS: Our study identifies FGF4 as a stress-responsive regulator of liver pathophysiology that acts through an FGFR4-AMPK-Caspase 6 signal pathway, shedding light on strategies for treating NAFLD and associated liver pathologies.

Paracrine FGFs target skeletal muscle to exert potent anti-hyperglycemic effects.

Several members of the FGF family have been identified as potential regulators of glucose homeostasis. We previously reported that a low threshold of FGF-induced FGF receptor 1c (FGFR1c) dimerization and activity is sufficient to evoke a glucose lowering activity. We therefore reasoned that ligand identity may not matter, and that besides paracrine FGF1 and endocrine FGF21, other cognate paracrine FGFs of FGFR1c might possess such activity. Indeed, via a side-by-side testing of multiple cognate FGFs of FGFR1c in diabetic mice we identified the paracrine FGF4 as a potent anti-hyperglycemic FGF. Importantly, we found that like FGF1, the paracrine FGF4 is also more efficacious than endocrine FGF21 in lowering blood glucose. We show that paracrine FGF4 and FGF1 exert their superior glycemic control by targeting skeletal muscle, which expresses copious FGFR1c but lacks ß-klotho (KLB), an obligatory FGF21 co-receptor. Mechanistically, both FGF4 and FGF1 upregulate GLUT4 cell surface abundance in skeletal muscle in an AMPKα-dependent but insulin-independent manner. Chronic treatment with rFGF4 improves insulin resistance and suppresses adipose macrophage infiltration and inflammation. Notably, unlike FGF1 (a pan-FGFR ligand), FGF4, which has more restricted FGFR1c binding specificity, has no apparent effect on food intake. The potent anti-hyperglycemic and anti-inflammatory properties of FGF4 testify to its promising potential for use in the treatment of T2D and related metabolic disorders.

Cell-cell communication through FGF4 generates and maintains robust proportions of differentiated cell types in embryonic stem cells.

During embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell-type proportioning despite variable initial conditions in the precursor cells, and the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterized. Here, we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range fibroblast growth factor 4 (FGF4) signal. We characterize the molecular and dynamical properties of the communication mechanism and show how it controls both robust cell-type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signaling that might operate in a range of developing tissues.

Fgf4 maintains Hes7 levels critical for normal somite segmentation clock function.

During vertebrate development, the presomitic mesoderm (PSM) periodically segments into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking only Fgf4 or Fgf8, which we previously demonstrated act redundantly to prevent PSM differentiation. Fgf8 is not required for somitogenesis, but Fgf4 mutants display a range of vertebral defects. We analyzed Fgf4 mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 maintains Hes7 levels and normal oscillatory patterns. To support our hypothesis that FGF4 regulates somitogenesis through Hes7, we demonstrate genetic synergy between Hes7 and Fgf4, but not with Fgf8. Our data indicate that Fgf4 is potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.

In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.

Expression of Fibroblast Growth Factor 4 in a Rat Model of Polydactyly of the Thumb Induced by Cytarabine.

BACKGROUND The aim of this study was to assess the expression and mechanisms of fibroblast growth factor 4 in polydactyly of the thumb induced by cytarabine. MATERIAL AND METHODS Rats were intraperitoneally injected with cytarabine at different gestation periods (12.5 days, 13.5 days, and 14.5 days) to establish a polydactyly of the thumb model. Then, the expression of FGF4 in polydactyly was studied by whole-mount in situ hybridization. We used hematoxylin & eosin stain and cartilage stain to investigate the development of the skeleton and tissues in the embryo. Pictures were taken to determine the general shape of the deformity, then X-rays were taken to detect bone distortion of the rats born with a congenital malformation. RESULTS In the experimental group (11.5 days, 12.5 days, 13.5 days, and 14.5 days), whole-mount in situ hybridization showed that the FGF4 expression at the tip of the embryonic limb bud was significantly increased compared with the control group and FGF4 was distributed in a wider range and lasted longer than in the control group (P<0.01). HE staining and cartilage staining showed that there was an extra metacarpal bone and a phalanx in the rats with polydactyly of the thumb (P<0.01). Images of the deformed limbs showed polydactyly and syndactyly of the thumb in the rats. Further X-ray examination revealed 1 extra metacarpal bone and 1 extra phalanx. CONCLUSIONS Cytarabine can induce polydactyly and syndactyly of the thumb in rats. In this process, cytarabine can induce the expression of FGF4 on the tip of the embryonic limb bud, which further leads to abnormal development of the embryonic limb bud and eventually causes a congenital deformity.

Proliferative Status in the Aqueous Humor of Eyes With Congenital Cataract.

PURPOSE: To measure the concentrations of growth factors in the aqueous humor of patients with congenital cataract and to investigate the biological effects of a selected cytokine (fibroblast growth factor 4 [FGF4]) on cell proliferation, migration, and transformation. METHODS: In the aqueous humor obtained from 55 eyes with congenital cataract and 55 eyes with age-related cataract, 40 growth factors were screened and selected cytokines were confirmed with enzyme-linked immunosorbent assays. After the addition of various concentrations of FGF4 (0, 2.5, 15, or 50 ng/mL) to the incubation medium, cellular functions were evaluated. RESULTS: The concentration of FGF4 was significantly higher in the aqueous humor of patients with congenital cataract than in that of patients with age-related cataract. The human SRA01/04 lens epithelial cell line was treated with FGF4 and the cell proliferation increased significantly both dose- and time-dependently. The wound healing assay and Transwell migration assay revealed a significant increase in the migration capacity of the SRA01/04 cell line treated with 15 or 50 ng/mL of FGF4 compared with that of control cells. The intensity of immunofluorescent staining for α-smooth muscle actin increased significantly in the SRA01/04 cell line when treated with FGF4. Cytoskeletal protein (F-actin) staining showed that changes of cell morphology were induced in primary lens epithelial cells by FGF4. CONCLUSIONS: This study provides a comprehensive profile of growth factors in congenital cataract. FGF4 induced cellular changes, and may have utility as a biomarker to predict the formation of visual axis opacification. [J Pediatr Ophthalmol Strabismus. 2020;57(3):159-168.].

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation.

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.

Simultaneous Derivation of Embryonic and Trophoblast Stem Cells from Mouse Blastocysts.

The formation of the blastocyst during mammalian development involves the segregation of two populations of cells with unequal potential: pluripotent cells of the inner cell mass (ICM) and multipotent cells of the trophectoderm (TE). ICM cells maintain the capacity to give rise to all cells represented in the organism, while TE cells, which represent the first lineage to emerge during development, are capable of differentiating into trophoblast lineages of the placenta. The ICM and TE are both essential for development. The ICM is genetically programmed to generate all cells of the embryo proper, while the TE forms extraembryonic trophoblast lineages and is required for implantation of the embryo and maternal-fetal exchange of nutrients and waste. Embryonic stem (ES) cells, which can be derived from the ICM of blastocysts in the presence of external signals such as LIF, can self-renewal indefinitely, and because they can differentiate into all cells of the organism, ES cells are a widely used in vitro model to study genetics and development. Trophoblast stem (TS) cells can be derived from the TE of blastocyst stage embryos in the presence of FGF4, and like ES cells, TS cells are also able to self-renew indefinitely. Because TS cells can differentiate into epithelial lineages of the trophoblast, TS cells are an ideal in vitro model to study the biology of the trophoblast. In this chapter, we describe protocols for simultaneous derivation of ES cells and TS cells from mouse blastocysts and culture conditions that promote self-renewal of hybrid ESC/TSC colonies. These protocols are sufficient for efficient derivation of hybrid ESC/TSC colonies.

Modulating cardiomyocyte and fibroblast interaction using layer-by-layer deposition facilitates synchronisation of cardiac macro tissues.

Maturation and synchronisation of heart cells, including cardiomyocytes and fibroblasts, are essential to develop functional biomimetic cardiac tissues for regenerative medicine and drug discovery. Synchronisation of cells in the biomimetic cardiac tissue requires the structural integrity and functional maturation of cardiomyocytes with other cell types. However, it is challenging to synchronise the beating of macroscale cardiac tissues and induce maturation of cardiomyocytes derived from stem cells. Here, we developed a simple assembly technology to modulate cell-cell interactions by combining layer-by-layer (LBL) deposition and centrifugation of cells with collagen type I to control cell-cell interactions for the preparation of cardiac macro tissues (CMTs). We found that maturation of cardiomyocytes in CMTs was largely enhanced by growth factors FGF-4 and ascorbic acid, but synchronisation of cardiac beating required LBL deposition of cardiomyocytes and cardiac fibroblasts in addition to the growth factors during the maturation process. Our findings have important implications because incorporation of cardiac fibroblasts into the cardiomyocyte layer is a prerequisite for synchronised beating of macroscale cardiac tissues in addition to growth factors to facilitate maturation of stem cell-derived cardiomyocytes.

B-Cell Activating Factor Enhances Hepatocyte-Driven Angiogenesis via B-Cell CLL/Lymphoma 10/Nuclear Factor-KappaB Signaling during Liver Regeneration.

B-cell activating factor (BAFF) is found to be associated with the histological severity of nonalcoholic steatohepatitis (NASH). BAFF was also found to have a protective role in hepatic steatosis via down regulating the expression of steatogenesis genes and enhancing steatosis in hepatocytes through BAFF-R. However, the roles of BAFF during liver regeneration are not well defined. In this study, C57/B6 mice with 70% partial hepatectomy were used as a liver regeneration model. BAFF expression was determined by enzyme immunoassay, and anti-BAFF-neutralizing antibodies were administered to confirm the effects of BAFF on liver regeneration. Western blotting, immunohistochemistry, and florescence staining determined the expression of B-cell CCL/lymphoma 10 (BCL10). The angiogenesis promoting capability was evaluated after the transfection of cells with siRNA targeting BCL10 expression, and the role of NF-κB was assessed. The results revealed that the BAFF and BCL10 levels were upregulated after partial hepatectomy. Treatment with anti-BAFF-neutralizing antibodies caused death in mice that were subjected to 70% partial hepatectomy within 72 h. In vitro, recombinant BAFF protein did not enhance hepatocyte proliferation; however, transfection with BCL10 siRNA arrested hepatocytes at the G2/M phase. Interestingly, conditioned medium from BAFF-treated hepatocytes enhanced angiogenesis and endothelial cell proliferation. Moreover, Matrix metalloproteinase-9 (MMP-9), Fibroblast growth factor 4 (FGF4), and Interleukin-8 (IL-8) proteins were upregulated by BAFF through BCL10/NF-κB signaling. In mice that were treated with anti-BAFF-neutralizing antibodies, the microvessel density (MVD) of the remaining liver tissues and liver regeneration were both reduced. Taken together, our study demonstrated that an increased expression of BAFF and activation of BCL10/NF-κB signaling were involved in hepatocyte-driven angiogenesis and survival during liver regeneration.

Regulation of the ERK signalling pathway in the developing mouse blastocyst.

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation occurs in ICM precursor cells, in differentiated primitive endoderm (PrE) cells as well as in the mature, formative state epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative-feedback loops of the FGF pathway, are differently regulated. Whereas DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into the PrE while protecting the Epi identity from autocrine signalling.

Critical role of the fibroblast growth factor signalling pathway in Ewing's sarcoma octamer-binding transcription factor 4-mediated cell proliferation and tumorigenesis.

Certain bone and soft tissue (BST) tumours harbour a chromosomal translocation [t(6;22)(p21;q12)], which fuses the Ewing's sarcoma (EWS) gene at 22q12 with the octamer-binding transcription factor 4 (Oct-4) gene at 6p21, resulting in the chimeric EWS-Oct-4 protein that possesses high transactivation ability. Although abnormal activation of signalling pathways can lead to human cancer development, the pathways underlying these processes in human BST tumours remain poorly explored. Here, we investigated the functional significance of fibroblast growth factor (FGF) signalling in human BST tumours. To identify the gene(s) involved in the FGF signalling pathway and potentially regulated by EWS-Oct-4 (also called EWS-POU5F1), we performed RNA-Seq analysis, electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and xenograft assays. Treating GBS6 or ZHBTc4 cells-expressing EWS-Oct-4 with the small molecule FGF receptor (FGFR) inhibitors PD173074, NVPBGJ398, ponatinib, and dovitinib suppressed cellular proliferation. Gene expression analysis revealed that, among 22 Fgf and four Fgfr family members, Fgf-4 showed the highest upregulation (by 145-fold) in ZHBTc4 cells-expressing EWS-Oct-4. Computer-assisted analysis identified a putative EWS-Oct-4-binding site at +3017/+3024, suggesting that EWS-Oct-4 regulates Fgf-4 expression in human BST tumours. Fgf-4 enhancer constructs showed that EWS-Oct-4 transactivated the Fgf-4 gene reporter in vitro, and that overexpression of EWS-Oct-4 stimulated endogenous Fgf-4 gene expression in vivo. Finally, PD173074 significantly decreased tumour volume in mice. Taken together, these data suggest that FGF-4 signalling is involved in EWS-Oct-4-mediated tumorigenesis, and that its inhibition impairs tumour growth in vivo significantly.

Suppression of TGF-ß and ERK Signaling Pathways as a New Strategy to Provide Rodent and Non-Rodent Pluripotent Stem Cells.

Stem cells are unspecialized cells and excellent model in developmental biology and a promising approach to the treatment of disease and injury. In the last 30 years, pluripotent embryonic stem (ES) cells were established from murine and primate sources, and display indefinite replicative potential and the ability to differentiate to all three embryonic germ layers. Despite large efforts in many aspects of rodent and non-rodent pluripotent stem cell culture, a number of diverse challenges remain. Natural and synthetic small molecules (SMs) strategy has the potential to overcome these hurdles. Small molecules are typically fast and reversible that target specific signaling pathways, epigenetic processes and other cellular processes. Inhibition of the transforming growth factor-ß (TGF-ß/Smad) and fibroblast growth factor 4 (FGF4)/ERK signaling pathways by SB431542 and PD0325901 small molecules, respectively, known as R2i, enhances the efficiency of mouse, rat, and chicken pluripotent stem cells passaging from different genetic backgrounds. Therefore, the application of SM inhibitors of TGF-ß and ERK1/2 with leukemia inhibitory factor (LIF) allows the cultivation of pluripotent stem cells in a chemically defined condition. In this review, we discuss recently emerging evidence that dual inhibition of TGF-ß and FGF signaling pathways plays an important role in regulating pluripotency in both rodent and non-rodent pluripotent stem cells.

Gain of FGF4 is a frequent event in KIT/PDGFRA/SDH/RAS-P WT GIST.

Gastrointestinal stromal tumors (GIST) lacking mutations in KIT/PDGFRA or RAS pathways and retaining an intact SDH complex are usually referred to as KIT/PDGFRA/SDH/RAS-P WT GIST or more simply quadruple WT GIST (~5% of all GIST). Despite efforts made, no recurrent genetic event in quadruple WT GIST has been identified so far. To further investigate this disease, we performed high throughput copy number analysis on quadruple WT GIST specimens identifying a recurrent focal gain in band 11q13.3 (involving FGF3/FGF4) in 6/8 cases. This event was not found in the other molecular GIST subgroups. FGF3/FGF4 duplication was associated with high expression of FGF4, both at mRNA and protein level, a growth factor normally not expressed in adult tissues or in KIT/PDGFRA-mutated GIST. FGFR1 was found to be the predominant FGF receptor expressed and phosphorylation of AKT was detected, suggesting that a FGF4-FGFR1 autocrine loop could stimulate downstream signaling in quadruple WT GIST. Together with the recent reports of quadruple WT cases carrying FGFR1 activating alterations, these findings strengthen the hypothesis of a potential involvement of FGFR pathway deregulation in quadruple WT GIST, which may represent a rationale for novel therapeutic approaches.

Induced Pluripotent Stem Cells Reprogrammed with Three Inhibitors Show Accelerated Differentiation Potentials with High Levels of 2-Cell Stage Marker Expression.

Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3ß (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.