FGF2 Treatment Research Articles

Expression profile of FGF receptors in preimplantation ovine embryos and the effect of FGF2 and PD173074

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are increasingly recognized as important regulators of embryo development in mammals. This study investigated the importance of FGF signaling during in vitro development of ovine embryo. The mRNAs of four FGFR subtypes were detected throughout preimplantation development of in vitro fertilized (IVF) embryos, peaked in abundance at the morula stage, and decreased significantly at the blastocyst stage. To gain insight into the role of these mRNAs in embryo development, IVF embryos were cultured in the presence of FGF2 (100 or 500 ng/ml: beginning from days 1 or 4 to 7) or PD173074 (1 µM: beginning from days 1 to 7) as usual treatments for activation or inhibition of FGFRs, respectively. FGF2-supplementation did not affect the percentage of embryos that developed to the blastocyst, blastocyst cell count and the proportion of cells allocated in inner cell mass (ICM) and trophectoderm (TE) compared to control (p > 0.05). Also, increasing the dosage or duration of FGF2 treatment did not significantly alter blastocyst yield or differential cell count (p > 0.05). PD173074-mediated inhibition of FGFRs did not significantly affect blastocyst yield (p > 0.05). Assessment of expression profiles of lineage-associated markers revealed that FGF2 (500 ng/ml) supplementation: (i) significantly increased expression of putative hypoblast marker (GATA4), (ii) significantly decreased expression of putative epiblast (EPI) marker (NANOG) and (iii) did not change TE markers (CDX2 and IFNT) and pluripotency makers (OCT4, SOX2 and REX1). In summary, FGF2-mediated activation of FGFRs may promote a switch in transcriptional profile of ovine ICM from EPI- to hypoblast-associated gene expression.

A Brn2–Zic1 axis specifies the neuronal fate of retinoic-acid-treated embryonic stem cells

Mouse embryonic stem cells (ESCs) treated with all-trans retinoic acid differentiate into a homogenous population of glutamatergic neurons. Although differentiation is initiated through activation of target genes by the retinoic acid receptors, the downstream transcription factors specifying neuronal fate are less well characterised. Here, we show that the transcription factor Brn2 (also known as Pou3f2) is essential for the neuronal differentiation programme. By integrating results from RNA-seq following Brn2 silencing with results from Brn2 ChIP-seq, we identify a set of Brn2 target genes required for the neurogenic programme. Further integration of Brn2 ChIP-seq data from retinoic-acid-treated ESCs and P19 cells with data from ESCs differentiated into neuronal precursors by Fgf2 treatment and that from fibroblasts trans-differentiated into neurons by ectopic Brn2 expression showed that Brn2 occupied a distinct but overlapping set of genomic loci in these differing conditions. However, a set of common binding sites and target genes defined the core of the Brn2-regulated neuronal programme, among which was that encoding the transcription factor Zic1. Small hairpin RNA (shRNA)-mediated silencing of Zic1 prevented ESCs from differentiating into neuronal precursors, thus defining a hierarchical Brn2-Zic1 axis that is essential to specify neural fate in retinoic-acid-treated ESCs.

Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway.

Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future.

Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells

Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

Abstract 493: Syndesomes: An Syndecan-4 Based Therapeutic for Effective Revascularization in Peripheral Ischemia in Diabetes

Previous research has attempted to use growth factor proteins and genes to revascularize ischemic tissues but none of the methods have found complete success in clinical trials. We have demonstrated the loss of co-receptors like syndecan-4 due to long-term diseased state and enhanced neovascularization of ischemic tissue due to co-delivery of syndesomes (syndecan-4 proteoliposomes) with FGF-2 in normal healthy rats. In this study, we used syndesomes to enhance revascularization in the ischemic hind limb of a growth factor resistant mouse model with diabetes, obesity and hyperlipidemia. We used ob/ob mice fed with high fat diet for 10 weeks. We induced ischemia through femoral artery ligation and implanted alginate beads containing treatments into the surgical site. The recovery of perfusion in the mice was tracked using laser speckle imaging. After 14 days, the ischemic limbs of the mice treated with FGF-2 with syndesomes had significantly higher perfusion compared to the FGF-2 alone group (A, B). Morphometric analysis on sections from the calf and thigh muscle of the mice demonstrated increased ischemic changes in the ischemic limbs of the FGF-2 alone group. Immunostaining for von Willebrand factor revealed increased neovascularization corresponding with the increase perfusion measured in the FGF-2 with syndesomes group (C, D). Immunostaining for pro-inflammatory M1 macrophage marker CD86 showed similar levels in both treatment groups (E, F). However, immunostaining for pro-wound healing M2 macrophage marker revealed significantly higher levels in the syndesome with FGF-2 treatment group (G, H). Taken together, these preliminary studies support that co-delivery of syndecan-4 with FGF-2 significantly enhances revascularization in the ischemic hind limb in a clinically relevant diseased mouse model. Our treatment restores the signaling pathway components that are lost due to diseased state causing tissues to become resistant to growth factor therapies.

Xenoestrogens disrupt type 2 collagen expression in chondrocytes in vitro.

Purpose: In recent years chemicals with endocrine properties, called Endocrine Disrupting Compounds (EDC), have become a topic of scientific and public discussion. In the field of osteoarthritic pathologies, EDC compounds that mimic estrogenic agonists or antagonists are of major concern, because of the central role of estrogens in skeletal developmental processes (ref). EDC are acting on their target genes upon binding the aryl hydrocarbon receptor (AhR). During the ten past years, interactions between estrogen receptor (ER) and some xenobiotic-activated AhR have been well demonstrated. Very few studies have been reported on EDC effects on bone or cartilage. Our laboratory recently studied the effects of low doses and cocktail xenoestrogens on rat cartilage development in vivo, by using a rodent gestational/lactational model (T.A. Auxietre, et al, 2014). We observed transient modifications of caudal vertebral body associated with a decrease in growth plate cartilage thickness with greater impact on the hypertrophic chondrocyte zone. We inferred from these data that the tested compounds could interfere with the dynamic of chondrogenic differentiation and/or maturation processes, possibly by impacting type 2 collagen (Col2), one of the the major components of cartilage that assures cartilage function. The present work was aimed to study the in vitro effects of two xenoestrogens (genistein, G; bisphenol A, BPA) and one anti-androgen (vinclozolin, V) and its metabolite M2 on Col2 expression in two different models of cultured chondrocytes: during the process of chondrogenic induction and in mature chondrocytes. Methods: A murine stem cell line inducible towards chondrogenesis (C1) was used for dynamic studies of differentiation using chondrogenic and non-chondrogenic markers. In parallel, post-natal murine chondrocytes were used for steady-state investigations in differentiated chondrocytes in primary culture (P0) or after dedifferentiation by passages (P3) or FGF2 treatment. Biological markers were studied at the mRNA (qPCR) and protein (western blot) levels. COL2A protein expression was evaluated by western blot and immunocytochemistry by using an antibody which only recognizes the specific COL2A N-terminal propeptide. Fulvestrant and SB 203580 were used respectively as specific inhibitors of ER or p38MEK pathways. Implication of Tia1-splicing was shown after cell transfection with specific anti-Tia1 siRNAs. Results: Vinclozolin was practically inactive. M2, alone or combined with G or BPA, modified the dynamic of Col2A immature isoform of COL2 during chondrogenic induction. These compounds extended the basal expression of COL2A and delayed its replacement by the mature isoform COL2B in C1 cells. This effect was dose dependent with maximum at 10-6M. In post natal chondrocytes, COL2A expression increased with cell dedifferentiation or FGF2 treatment. EDC were inactive on differentiated chondrocytes while COL2A doubled upon M2, G and BPA addition in dedifferentiated cells. Estrogen receptor (ER) and the p38-MEK pathway were involved. As these effects were only partly transcriptional, we investigated and found an effect of FGF2 on Tia-1 splice protein expression. EDC showed no effects on other chondrogenic markers (SOX9, Aggrecan, Col10α1) nor non-chondrogenic markers. Conclusions: These data showed that xenosestrogens as well as the vinclozolin metabolite M2 modified the rate of early chondrogenic differentiation by transiently maintaining COL2A expression. Our hypothesis is that COL2A induced persistence, might provoke matrix weakening in the long term and/or sequester growth factors, eventually modifying the course of cartilage degenerative diseases such as osteoarthritis. EDC also modified COL2A expression in dedifferentiated chondrocytes such as found in aging or osteoarthritic tissues. These data are in accordance with the strong Col2A expression reported in OA cartilage but its role remains to be studied.

Induction of endogenous interleukin 1 alpha limits to one cycle the proliferative response to FGF

We found that transient FGF stimulation of various cell types results in the novel phenomenon termed “FGF memory” – a sustained blockage of efficient proliferative response to FGF, PDGF or EGF. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates the proinflammatory NFkappaB signaling, which is also critical for FGF memory formation. The search of potential FGF‐induced mediators of FGF memory revealed that FGF induces a sustained expression of the inflammatory cytokine IL1alpha, and this induction is abolished by HDAC inhibitors. Similarly to FGF pretreatment, transient cell treatment with recombinant IL1alphainhibits the proliferative response to further FGF stimulation. However, cells pretreated with FGF or IL1alpha exhibit an enhanced restructuring of actin cytoskeleton and migration in response to FGF. IL1alpha‐prestimulated cells respond to FGF treatment by ERK1/2 phosphorylation and expression of cyclin D1, but not of cyclin A. Both IRAP, a specific inhibitor of IL1 receptor, and neutralizing anti‐IL1alphaantibodies prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. Thus, FGF memory is mediated by the endogenous IL1alpha. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound‐induced hyperplasia.

Sensitivity of Melanoma Cells to EGFR and FGFR Activation but Not Inhibition is Influenced by Oncogenic BRAF and NRAS Mutations.

BRAF and NRAS are the two most frequent oncogenic driver mutations in melanoma and are pivotal components of both the EGF and FGF signaling network. Accordingly, we investigated the effect of BRAF and NRAS oncogenic mutation on the response to the stimulation and inhibition of epidermal and fibroblast growth factor receptors in melanoma cells. In the three BRAF mutant, two NRAS mutant and two double wild-type cell lines growth factor receptor expression had been verified by qRT-PCR. Cell proliferation and migration were determined by the analysis of 3-days-long time-lapse videomicroscopic recordings. Of note, a more profound response was found in motility as compared to proliferation and double wild-type cells displayed a higher sensitivity to EGF and FGF2 treatment when compared to mutant cells. Both baseline and induced activation of the growth factor signaling was assessed by immunoblot analysis of the phosphorylation of the downstream effectors Erk1/2. Low baseline and higher inducibility of the signaling pathway was characteristic in double wild-type cells. In contrast, oncogenic BRAF or NRAS mutation did not influence the response to EGF or FGF receptor inhibitors in vitro. Our findings demonstrate that the oncogenic mutations in melanoma have a profound impact on the motogenic effect of the activation of growth factor receptor signaling. Since emerging molecularly targeted therapies aim at the growth factor receptor signaling, the appropriate mutational analysis of individual melanoma cases is essential in both preclinical studies and in the clinical trials and practice.

A Brn2-Zic1 axis specifies the neuronal fate of retinoic-acid-treated embryonic stem cells.

Mouse embryonic stem cells (ESCs) treated with all-trans retinoic acid differentiate into a homogenous population of glutamatergic neurons. Although differentiation is initiated through activation of target genes by the retinoic acid receptors, the downstream transcription factors specifying neuronal fate are less well characterised. Here, we show that the transcription factor Brn2 (also known as Pou3f2) is essential for the neuronal differentiation programme. By integrating results from RNA-seq following Brn2 silencing with results from Brn2 ChIP-seq, we identify a set of Brn2 target genes required for the neurogenic programme. Further integration of Brn2 ChIP-seq data from retinoic-acid-treated ESCs and P19 cells with data from ESCs differentiated into neuronal precursors by Fgf2 treatment and that from fibroblasts trans-differentiated into neurons by ectopic Brn2 expression showed that Brn2 occupied a distinct but overlapping set of genomic loci in these differing conditions. However, a set of common binding sites and target genes defined the core of the Brn2-regulated neuronal programme, among which was that encoding the transcription factor Zic1. Small hairpin RNA (shRNA)-mediated silencing of Zic1 prevented ESCs from differentiating into neuronal precursors, thus defining a hierarchical Brn2-Zic1 axis that is essential to specify neural fate in retinoic-acid-treated ESCs.

PV.1 induced by FGF-Xbra functions as a repressor of neurogenesis in Xenopus embryos

During Xenopus early development, FGF signaling is involved in mesoderm formation and neurogenesis by modulating various signaling cascades. FGF-MAPK signaling induces Xbra expression, which maintains mesodermal fate through an autocatalytic-loop. Interestingly, previous reports have demonstrated that basic FGF (bFGF) treatment alone does not induce neurogenesis in ectodermal explants, even though FGF signaling inhibits BMP signaling via phosphorylation in Smad1 linker region. In addition, the overexpression of dominantnegative Xbra induces neurogenesis in ectodermal explants. However, the detailed mechanism underlying these phenomena has not yet been clarified. In this work, we showed that bFGF-Xbra signaling increased the PV.1 expression. DN-Xbra was found to decrease PV.1 expression, and the co-injection of PV.1 with DN-Xbra reduced neurogenesis in ectodermal explants. Furthermore, the knockdown of PV.1 induced neurogenesis in bFGF-treated ectodermal explants. Taken together, our results demonstrate that FGF-Xbra signaling induces PV.1 expression and that PV.1 functions as a neural repressor in the FGF-treated ectoderm. [BMB Reports 2014; 47(12): 673-678]

Decreased anti-regenerative effects after spinal cord injury in spry4−/− mice

Previously, we have demonstrated a role for fibroblast growth factor (Fgf) in spinal cord regeneration in both zebrafish and mouse. We have shown that exogenous Fgf2 treatment attenuates astrocytic gliosis and induces glia cells to become progenitors that undergo neurogenesis as well as differentiating into bipolar astrocytes that support axonal regeneration (Goldshmit et al., 2012, 2014). One of the downstream signaling target genes of Fgf is spry4, which acts as a feedback inhibitor for Fgf signaling. In this study we examined the effects of increased endogenous Fgf signaling, in spry4−/− mice, on the early events that occur after spinal cord injury (SCI). We demonstrate that in spry4−/− mice inflammatory responses, such as tumor necrosis factor α (TNFα) secretion and macrophage/neutrophil invasion into the lesion site are reduced. In addition, astrocytic gliosis is attenuated and neuronal survival is increased. These results further support a pro-regenerative role of Fgf after SCI, and suggest that increased endogenous Fgf signaling after SCI may contribute to functional recovery and therefore presents this pathway as a target for new therapy development.

Fibroblast growth factor 10 protects neuron against oxygen–glucose deprivation injury through inducing heme oxygenase-1

Fibroblast growth factors (FGFs) are a family of structurally related heparin-binding proteins with diverse biological functions. FGFs participate in mitogenesis, angiogenesis, cell proliferation, development, differentiation and cell migration. Here, we investigated the potential effect of FGF10, a member of FGFs, on neuron survival in oxygen–glucose deprivation (OGD) model. In primary cultured mouse cortical neurons upon OGD, FGF10 treatment (100 and 1000ng/ml) attenuated the decrease of cell viability and rescued the LDH release. Tuj-1 immunocytochemistry assay showed that FGF10 promoted neuronal survival. Apoptosis assay with Annexin V+PI by flow cytometry demonstrated that FGF10 treatment reduced apoptotic cell proportion. Moreover, immunoblotting showed that FGF10 alleviated the cleaved caspase-3 upregulation caused by OGD. FGF10 treatment also depressed the OGD-induced increase of caspase-3, -8 and -9 activities. At last, we found FGF10 triggered heme oxygenase-1 (HO-1) protein expression rather than hypoxia-inducible factor-1 (HIF-1), AMP-activated protein kinase (AMPK) signaling and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling. Knockdown of HO-1 by siRNA partly abolished the neuroprotection of FGF10 in OGD model. In summary, our observations provide the first evidence for the neuroprotective function of FGF10 against ischemic neuronal injury and suggest that FGF10 may be a promising agent for treatment of ischemic stroke.

A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.

In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.

Synergistic induction of early stage of bone formation by combination of recombinant human bone morphogenetic protein-2 and epidermal growth factor.

This study evaluates whether the combination of the rhBMP-2 and various types of growth factors including EGF, FGF, PDGF and VEGF increases osteoinductivity compared to the single use of rhBMP-2 through in vitro and in vivo study. Cultured human MSCs were treated with rhBMP-2 only or in combination with growth factors. For in vivo evaluation, rhBMP-2 only or with growth factors was implanted into the calvarial defect made on SD rats. Both EGF and PDGF significantly increased both ALP activity and expression level in hMSCs when treated in combination with rhBMP-2 at 3 and 7 days of differentiation and significantly raised the accumulation of the calcium at day 14. Furthermore, micro-CT scanning revealed that the EGF an FGF groups show significantly increased new bone surface ratio compared to the rhBMP-2 only group and, the EGF treatment significantly up regulated percent bone volume and trabecular number at two weeks after the surgery. VEGF treatment also significantly raised trabecular number and FGF treatment significantly increased the trabecular thickness. Histological examination revealed that the EGF combination group showed enhanced bone regeneration than the rhBMP-2 only group two weeks after the implantation. Even though the treatment of rhBMP-2 with PDGF and FGF failed to show enhanced osteogenesis in vitro and in vivo simultaneously, these results suggest that the positive effect of the combination of EGF and rhBMP-2 is expected to induce the bone formation earlier compared to the single use of rhBMP-2 in vitro and in vivo. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.

Immunohistochemical localization of fibroblast growth factor-2 in the sheep ovary and its effects on pre-antral follicle apoptosis and development in vitro.

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7days in control medium (α-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200ng/ml FGF-2 (p<0.05). However, the percentage of apoptotic cells only decreased (p<0.05) in ovarian tissues cultured in 1 or 10ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.

Transitory FGF Treatment Results in the Long‐Lasting Suppression of the Proliferative Response to Repeated FGF Stimulation

FGF applied as a single growth factor to quiescent mouse fibroblasts induces a round of DNA replication, however continuous stimulation results in arrest in the G1 phase of the next cell cycle. We hypothesized that FGF stimulation induces the establishment of cell memory, which prevents the proliferative response to repeated or continuous FGF application. When a 2-5 days quiescence period was introduced between primary and repeated FGF treatments, fibroblasts failed to efficiently replicate in response to secondary FGF application. The establishment of "FGF memory" during the first FGF stimulation did not require DNA synthesis, but was dependent on the activity of FGF receptors, MEK, p38 MAPK and NFκB signaling, and protein synthesis. While secondary stimulation resulted in strongly decreased replication rate, we did not observe any attenuation of morphological changes, Erk1/2 phosphorylation and cyclin D1 induction. However, secondary FGF stimulation failed to induce the expression of cyclin A, which is critical for the progression from G1 to S phase. Treatment of cells with a broad range histone deacetylase inhibitor during the primary FGF stimulation rescued the proliferative response to the secondary FGF treatment suggesting that the establishment of "FGF memory" may be based on epigenetic changes. We suggest that "FGF memory" can prevent the hyperplastic response to cell damage and inflammation, which are associated with an enhanced FGF production and secretion. "FGF memory" may present a natural obstacle to the efficient application of recombinant FGFs for the treatment of ulcers, ischemias, and wounds.

Fgf2 improves functional recovery—decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury

ObjectivesA major impediment for recovery after mammalian spinal cord injury (SCI) is the glial scar formed by proliferating reactive astrocytes. Finding factors that may reduce glial scarring, increase neuronal survival, and promote neurite outgrowth are of major importance for improving the outcome after SCI. Exogenous fibroblast growth factor (Fgf) has been shown to decrease injury volume and improve functional outcome; however, the mechanisms by which this is mediated are still largely unknown.MethodsIn this study, Fgf2 was administered for 2 weeks in mice subcutaneously, starting 30 min after spinal cord hemisection.ResultsFgf2 treatment decreased the expression of TNF-a at the lesion site, decreased monocyte/macrophage infiltration, and decreased gliosis. Fgf2 induced astrocytes to adopt a polarized morphology and increased expression of radial markers such as Pax6 and nestin. In addition, the levels of chondroitin sulfate proteoglycans (CSPGs), expressed by glia, were markedly decreased. Furthermore, Fgf2 treatment promotes the formation of parallel glial processes, “bridges,” at the lesion site that enable regenerating axons through the injury site. Additionally, Fgf2 treatment increased Sox2-expressing cells in the gray matter and neurogenesis around and at the lesion site. Importantly, these effects were correlated with enhanced functional recovery of the left paretic hind limb.ConclusionsThus, early pharmacological intervention with Fgf2 following SCI is neuroprotective and creates a proregenerative environment by the modulation of the glia response.

Uronyl 2-O sulfotransferase potentiates Fgf2-induced cell migration.

Fibroblast growth factor 2 (Fgf2) is involved in several biological functions. Fgf2 requires glycosaminoglycans, like chondroitin and dermatan sulfates (hereafter denoted CS/DS) as co-receptors. CS/DS are linear polysaccharides composed of repeating disaccharide units [-4GlcUAb1-3-GalNAc-b1-] and [-4IdoUAa1-3-GalNAc-b1-],which can be sulfated. Uronyl 2-O-sulfotransferase (Ust)introduces sulfation at the C2 of IdoUA and GlcUA resulting inover-sulfated units. Here, we investigated the role of Ust-mediated CS/DS 2-O sulfation in Fgf2-induced cell migration. We found that CHO-K1 cells overexpressing Ust contain significantly more CS/DS2-O sulfated units, whereas Ust knockdown abolished CS/DS 2-O sulfation. These structural differences in CS/DS resulted in altered Fgf2 binding and increased phosphorylation of ERK1/2 (also known as MAPK3 and MAPK1, respectively). As a functional consequence of CS/DS 2-O sulfation and altered Fgf2 binding, cell migration and paxillin activation were increased. Inhibition of sulfation, knockdown of Ust and inhibition of FgfR resulted in reduced migration. Similarly, in 3T3 cells Fgf2 treatment increased migration, which was abolished by Ust knockdown. The proteoglycan controlling the CHO migration was syndecan 1. Knockdown of Sdc1 in CHO-K1 cells overexpressing Ust abolished cell migration.We conclude that the presence of distinctly sulfated CS/DS can tune the Fgf2 effect on cell migration.

Perlecan is required for FGF-2 signaling in the neural stem cell niche

In the adult subventricular zone (neurogenic niche), neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

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.