Fibroblast Growth Factor-2 Protein Levels Research Articles

Estrogen, via ESR2 receptor, prevents oxidative stress-induced Müller cell death and stimulates FGF2 production independently of NRF2, attenuating retinal degeneration

In this study, we aimed to investigate the effects of the deficient antioxidative gene, nuclear factor-erythroid 2-related factor 2 (Nrf2), on 17β-estradiol (E2)-mediated oxidative stress response, with a specific focus on growth factor production and cell death in Müller cells and retinal tissue. Administration of hydrogen peroxide (H2O2) reduced the viability of Müller cells derived from Nrf2 wild-type (WT) and knockout (KO) mice. However, this effect was more significant in the KO cells than in the WT cells. Pretreatment with E2 inhibited H2O2-induced cell death in both Nrf2 WT and KO Müller cell genotypes. Small interfering RNA-mediated gene silencing of estrogen receptor 2 (Esr2) attenuated the cell survival-promoting activity of E2 in Nrf2 KO Müller cells, while other identified estrogen receptors, Esr1 or G protein-coupled estrogen receptor 1 (Gper1), had no effect. Western blotting revealed higher ESR2 expression levels in Nrf2 KO cells than in WT Müller cells. Conditioned media from E2-and H2O2-treated Nrf2 WT or KO Müller cells enhanced the dissociated retinal cell viability compared with H2O2-treated cells. Both quantitative reverse-transcription polymerase chain reaction assay (qRT-PCR) and enzyme-linked immunosorbent assay exhibited a significant increase in fibroblast growth factor 2 (FGF2) expression levels in E2-and H2O2-treated Nrf2 WT and KO Müller cells compared to those in E2-treated cells. In vivo, administration of N-methyl-N-nitrosourea (MNU) reduced the thickness and cell density of the outer nuclear layer (ONL) in Nrf2 KO mice and enhanced the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the ONL. However, E2 administration attenuated these defects in MNU-treated mice. Concomitant administration of MNU and E2 enhanced FGF2 protein levels in retinal lysates of Nrf2 KO mice. In conclusion, E2 demonstrated a significant role in preventing oxidative stress-induced retinal cell death by stimulating FGF2 production in Müller cells, independent of the Nrf2 gene. Based on these findings, we anticipate that exogenous administration of estrogens or ESR2-selective agonists could aid in treating patients with oxidative stress-related retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.

Upregulation of fibroblast growth factor 2 contributes to endometriosis through SPRYs/DUSP6/ERK signaling pathway

Previous studies report that fibroblast growth factor 2 (FGF2) modulates Sproutys (SPRYs)/dual specificity phosphatase 6 (DUSP6)/extracellular signal-regulated kinase (ERK) signaling pathway in endometrial glandular epithelial cells. However, its role in endometriosis remains unclear. The expression patterns and localization of related proteins in endometrium patients’ samples were determined using quantitative reverse transcription PCR, Western blotting, and immunohistochemistry, respectively. Human endometrial stromal cells (HESCs) were isolated and transfected with small interfering RNA (siRNA) targeting FGF2 (FGF2-siRNA). Cell viability was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It was found that FGF2 mRNA and protein levels were increased in the ectopic endometrium, whilst the mRNA and protein levels of SPRYs/DUSP6/ERK signaling pathway related-genes were dysregulated. Spearman’s rank correlation analysis revealed a negative correlation between FGF2 and SPRYs/DUSP6 signaling pathway-related proteins. In vitro study demonstrated that FGF2 silencing suppressed cell proliferation. Our results suggest that FGF2 upregulation might contribute to endometriosis via the regulation of the SPRYs/DUSP6/ERK signaling pathway.

Effect of FGF-AS Modulation on Neurogenesis in Rat Hippocampus

Fibroblast growth factor 2(FGF2) is a growth factor involved in the pathophysiology of major depression.FGF-antisense (FGF-AS), the natural antisense transcript of FGF2, is indicated in the regulation of FGF2 expression. We previously reported that hippocampal overexpression of FGF-AS resulted in depression-like behaviour, whereas FGF-AS knockdown had antidepressant and anxiolytic effects, which both were independent of changes in FGF2 protein levels. To have an insight into the mechanism of actions of FGF-AS protein, we investigated the effects of manipulations of FGF-AS expression on hippocampal neurogenesis, as there are many studies suggesting the involvement of neurogenesis in the regulation of affective behaviour.

Effects of fibroblast growth factor-2 on collateral artery formation in an ischemic myocardium promoted by isometric exercise

Objective To investigate the effect of fibroblast growth factor-2 (FGF-2) on the formation of collateral arteries in the remote myocardium promoted by the isometric exercise (IE). Methods Twenty-four male Sprague-Dawley rats weighing (200±20)g were randomized into a sham operation group (SO), a myocardial ischemia group (MI), an isometric exercise group (IE) and an FGF inhibitor group (Inhi-FGF), each of 6. Rats in the SO group were injected with saline subcutaneously for 2 weeks while those of the MI group were being injected with 10 mg/(kg·d) of isoproterenol subcutaneously to induce myocardial ischemia. Rats in group IE accepted isometric exercise and the same injections as group MI, while those in group Inhi-FGF were also given 100 mg/(kg·d) of formononetin intragastrically in addition to the group IE treatment. After 8 weeks of IE the myocardium of the rats′ left ventricles was resected. The relative collateral blood flow (RCBF) was measured using the microsphere method. The artery density and the number of smooth muscle cells were evaluated using immunohistochemical analysis. Western blotting was performed to assay the levels of FGF-2 protein and its receptor FGFR-1. Results Compared to the SO, MI and Inhi-FGF groups, significant increases in the RCBF, artery density, the number of smooth muscle cells and the relative levels of FGF-2 protein and FGFR-1 were observed in group IE. In the Inhi-FGF group the artery density, the number of smooth muscle cells and the relative protein level of FGF-2 were significantly lower than those of the MI group, but there were no significant differences in the RCBF or FGFR-1 levels between the two groups. The artery density had a positive linear correlation with the number of smooth muscle cells. The RCBF correlated with the artery density, the number of smooth muscle cells and the FGF-2 protein levels. Conclusion IE can promote the expression of FGF-2 and its acceptor, resulting in the formation of collateral arteries and better blood perfusion of an ischemic myocardium. Key words: Isometric exercise; Myocardial ischemia; Collateral artery formation; Fibroblast growth factors

MicroRNA‐195‐5p Regulates Osteogenic Differentiation of Periodontal Ligament Cells Under Mechanical Loading

Osteogenic differentiation and bone formation are tightly regulated by several factors, including microRNAs (miRNAs). However, miRNA expression patterns and function during mechanical loading-induced osteogenic differentiation of human periodontal ligament cells (PDLCs) remain unclear. Here, we investigated the differential expression of miRNA-195-5p in the periodontal tissues of mice under orthodontic mechanical loading and in primary human PDLCs exposed to a simulated tension strain. The miR-195-5p was observed to be down-regulated and negatively correlated with osteogenic differentiation. Overexpression of miR-195-5p significantly inhibited PDLC differentiation under cyclic tension strain (CTS), whereas the functional inhibition of miR-195-5p yielded an opposite effect. Further experiments confirmed that WNT family member 3A (WNT3A), fibroblast growth factor 2 (FGF2), and bone morphogenetic protein receptor-1A (BMPR1A), proteins important for osteogenic activity and stability, were direct targets of miR-195-5p. Mechanical loading increased the WNT3A, FGF2, and BMPR1A protein levels, while miR-195-5p inhibited WNT3A, FGF2, and BMPR1A protein expression. WNT, FGF, and BMP signaling were involved in osteogenic differentiation of PDLCs under CTS. Further study confirmed that reintroduction of WNT3A and BMPR1A can rescue the inhibition of miR-195-5p on osteogenic differentiation of PDLCs. Our findings are the first to demonstrate that miR-195-5p is a mechanosensitive gene that plays an important role in mechanical loading-induced osteogenic differentiation and bone formation.

Anxiolytic effects of muscarinic acetylcholine receptors agonist oxotremorine in chronically stressed rats and related changes in BDNF and FGF2 levels in the hippocampus and prefrontal cortex.

In depressive disorders, one of the mechanisms proposed for antidepressant drugs is the enhancement of synaptic plasticity in the hippocampus and cerebral cortex. Previously, we showed that the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine (Oxo) increases neuronal plasticity in hippocampal neurons via FGFR1 transactivation. Here, we aimed to explore (a) whether Oxo exerts anxiolytic effect in the rat model of anxiety-depression-like behavior induced by chronic restraint stress (CRS), and (b) if the anxiolytic effect of Oxo is associated with the modulation of neurotrophic factors, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF2), and phosphorylated Erk1/2 (p-Erk1/2) levels in the dorsal or ventral hippocampus and in the medial prefrontal cortex. The rats were randomly divided into four groups: control unstressed, CRS group, CRS group treated with 0.2mg/kg Oxo, and unstressed group treated with Oxo. After 21days of CRS, the groups were treated for 10days with Oxo or saline. The anxiolytic role of Oxo was tested by using the following: forced swimming test, novelty suppressed feeding test, elevated plus maze test, and light/dark box test. The hippocampi and prefrontal cortex were used to evaluate BDNF and FGF2 protein levels and p-Erk1/2 levels. Oxo treatment significantly attenuated anxiety induced by CRS. Moreover, Oxo treatment counteracted the CRS-induced reduction of BDNF and FGF2 levels in the ventral hippocampus and medial prefrontal cerebral cortex CONCLUSIONS: The present study showed that Oxo treatment ameliorates the stress-induced anxiety-like behavior and rescues FGF2 and BDNF levels in two brain regions involved in CRS-induced anxiety, ventral hippocampal formation, and medial prefrontal cortex.

Abstract B47: Curcumin C3 ® prevents ultraviolet B radiation-induced epidermal damage in JB6 cells and mouse skin via a Fibroblast growth factor-2-dependent manner

Abstract Background and Significance: Non-melanoma skin cancer (NMSC) is the most common skin cancer in the U.S., with over a million new cases anticipated annually. Cutaneous squamous cell carcinoma (SCC) is a more aggressive form of NMSC. Chronic exposure to solar ultraviolet (UVB) is the major etiologic factor causing skin cell damage, photoaging and malignant transformation leading to SCC. UVB is a carcinogen and our previous studies established oral administration of Curcumin C3 complex (C3), a natural occurring polyphenol compound mixture, delayed UVB-induced tumor onset and multiplicity, but not tumor progression. Accordingly, we sort to determine the preventative efficacy of C3® against UVB-induced photodamage using JB6 cells and SKH-1 hairless mouse epidermis on the earlier neoplastic events in the development of UVB-induced skin carcinogenesis. Methods: SKH-1 mice were either administered vehicle or C3 (100mg/kg/b.w) complex orally for 14 days followed by a single exposure to UVB radiation (180mj/cm2). Mice were euthanized 24 hours after UVB exposure and skin tissues and serum were collected. For in vitro studies, promoter–sensitive JB6 mouse epithelial cells were pre-treated with C3® for 2 h and the effects on UV-induced early molecular events were assessed. Results: C3® attenuated UVB-induced epidermal hyperplasia and significantly decreased epidermal and plasma levels of fibroblast growth factor-2 (FGF-2); an important mitogen implicated in proliferation and differentiation of skin keratinocytes. Interestingly, the effects of C3® complex on FGF-2 levels were accompanied with a significant reduction in UVB-induced Nuclear factor KappaB (NF-KB) activation; an important nuclear transcription factor for tumor promotion. Further, the decrease in NF-KB was associated with an inhibition of BCL2 expression to induce apoptosis. In vitro, pretreatment of JB6 cells with C3® significantly decreased UVB-induced proliferation with a corresponding decrease in FGF-2 mRNA and protein levels. Further, C3 complex inhibited NF-KB phosphorylation and increased Bax expression leading to cell apoptosis. Conclusion: Our findings underscore the importance of curcumin (C3) in preventing UVB-induced skin cancer. It provides new insight into curcumin's role through FGF-2 in modulating the pathogenesis of photo carcinogenesis. Citation Format: Alok R. Khandelwal, Arun Anandharaj, Oleksandr Ekshyyan, Lauren D. Saucier, Tara N. Moore-Medlin, Fleurette Abreo, Cherie-Ann O. Nathan. Curcumin C3 ® prevents ultraviolet B radiation-induced epidermal damage in JB6 cells and mouse skin via a Fibroblast growth factor-2-dependent manner. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B47.

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

Notch1 mediates visfatin-induced FGF-2 up-regulation and endothelial angiogenesis

Our aims were to determine the role of Notch1 in mediating visfatin-induced angiogenesis and to explore potential target genes involved. Inhibition of Notch signalling attenuated visfatin-induced angiogenesis in vitro, ex vivo, and in vivo. Visfatin increased γ-secretase activity, Notch1 cleavage and activation, and Hes1 gene induction. Visfatin also stimulated fibroblast growth factor-2 (FGF-2) gene expression in a Notch1-dependent manner. Enforced expression of active Notch1 intracellular domain increased FGF-2 protein levels and stimulated endothelial tube formation, whereas blocking Notch1 signalling or knockdown of Notch1 by small interfering RNA suppressed visfatin-induced FGF-2 up-regulation and angiogenesis. Reporter analysis of FGF-2 promoter revealed the presence of CSL (CBF-1, suppressor of hairless, LAG-1)-binding site, and chromatin immunoprecipitation analysis demonstrated the binding of Notch1-CSL complex to this site in response to visfatin. Our data provide the first example of Notch1-dependent endothelial FGF-2 induction by visfatin and of Notch1 activation in visfatin-stimulated endothelial angiogenesis, suggesting that the signalling axis of visfatin/Notch1/angiogenic factors like FGF-2 might be a valuable target for pathological angiogenesis.

Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation

We have previously demonstrated increased fibroblast growth factor-2 (FGF-2) expression in a lamb model of increased pulmonary blood flow secondary to congenital heart disease, which may contribute to the associated increases in pulmonary arterial muscularization. However, the mechanisms underlying these increases in FGF-2 expression remain to be identified. Initially, we found that exogenous FGF-2 increased endogenous FGF-2 promoter activity and protein levels in ovine pulmonary arterial smooth muscle cells (PASMC). Furthermore, we found that these increases in FGF-2 expression were mediated by increases in superoxide levels via NADPH oxidase activation. In addition, FGF-2-mediated increases in FGF-2 expression and PASMC proliferation were attenuated by inhibition of phosphatidylinositol 3-kinase, Akt, and NADPH oxidase. Increases in FGF-2 expression could be stimulated by other factors known to increase reactive oxygen species (ROS) signaling in PASMC (endothelin-1 and transforming growth factor-beta1), whereas antioxidants attenuated these increases. Deletion constructs localized the growth factor- and ROS-sensitive region within the proximal 103 bp of the FGF-2 promoter, and sequence analysis identified a putative hypoxia response element (HRE), a DNA binding site for the ROS-sensitive transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). Stabilization of HIF-1alpha increased FGF-2 promoter activity, whereas mutation of the putative HRE attenuated FGF-2-induced FGF-2 promoter activity. Furthermore, FGF-2 increased HIF-1alpha protein levels and consensus HRE promoter activity in PASMC via antioxidant-sensitive mechanisms. Thus we conclude that FGF-2 can stimulate its own expression in PASMC via NADPH oxidase-mediated activation of ROS-sensitive transcription factors, including HIF-1alpha. This positive feedback mechanism may contribute to pulmonary vascular remodeling associated with increased pulmonary blood flow.

Ovine endometrial expression of fibroblast growth factor (FGF) 2 and conceptus expression of FGF receptors during early pregnancy

In ruminants, conceptus development beyond the blastocyst state requires input from uterine-derived factors. Fibroblast growth factor 2 (FGF2) is expressed by the bovine endometrium throughout the estrus cycle and early pregnancy and stimulates trophectoderm expression of interferon-tau, the maternal recognition of pregnancy factor in ruminants. The objective of this study was to examine the expression of FGF2 in ovine endometrium and peri-attachment conceptuses and FGF receptors (FGFR) in conceptuses. FGF2 mRNA was present in the ovine endometrium with specific localization within the luminal and glandular epithelium. No pregnancy-dependent changes in endometrial FGF2 mRNA abundance were detected until placental attachment was well underway. FGF2 protein was detected in the uterine lumen throughout the estrous cycle and early pregnancy. Concentrations of luminal FGF2 protein did not differ based on pregnancy status. However, uterine luminal FGF2 protein levels increased at days 12–13 after estrus in both cyclic and pregnant ewes. Ovine conceptuses collected at days 14–19 after mating contained transcripts for FGF2 and FGFR types 1, 2 and 3. In summary, FGF2 is expressed by the ovine endometrium and conceptus during early pregnancy, and peri-attachment conceptuses possess several FGFR types. Concentrations of FGF2 protein in the uterine lumen increase coincident with the initiation of pregnancy recognition in ewes. These observations support the concept that FGF2 and potentially other FGFs may affect conceptus development and/or gene expression during early pregnancy in ruminants.

Fibroblast Growth Factor-2 Expression Is Altered in Lambs With Increased Pulmonary Blood Flow and Pulmonary Hypertension

A lamb model of pulmonary hypertension, developed by inserting an aortopulmonary vascular graft (shunt), displays vascular remodeling and increased pulmonary blood flow characteristic of children with congenital heart disease. The purpose of this study was to determine whether expression of fibroblast growth factor-2 (FGF-2), a smooth muscle cell mitogen, is altered in shunt lambs. FGF-2 mRNA and protein levels were increased in lung tissue extracts from shunt lambs at 4 wk of age relative to age-matched controls (p < 0.05). FGF-2 protein levels were also increased in the pulmonary arteries and serum of shunt lambs (p < 0.05). Pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were isolated from 4 wk-old lambs and subjected to cyclic stretch and laminar shear stress to mimic increased pulmonary blood flow. Stretch and shear increased FGF-2 promoter activity, and intracellular and extracellular FGF-2 protein levels in both cell types (p < 0.05). Exogenous FGF-2 stimulated PASMC proliferation at levels detected in the extracellular medium of sheared cells (p < 0.05). Elevated FGF-2 signaling by PASMC and PAEC exposed to increased pulmonary blood flow may play a role in the pulmonary vascular remodeling associated with the shunt model of pulmonary hypertension secondary to congenital heart disease.

Alterations in Seizure Susceptibility and in Seizure‐induced Plasticity after Pharmacologic and Genetic Manipulation of the Fibroblast Growth Factor‐2 System

The adult brain undergoes activity-dependent plastic modifications during pathologic processes that are reminiscent of those observed during development. For example, seizures induce neuronal loss, neurogenesis, axonal and dendritic sprouting, gliosis, and circuit remodeling. Neurotrophic factors and fibroblast growth factor-2 (FGF-2), in particular, are well-known mediators in each of these cellular events. The aim of this minireview is to summarize and discuss the data supporting the idea that FGF-2 may be involved in seizure generation and in their sequelae. We used epilepsy models of kainate and kindling, with FGF-2 knockout mice and FGF-2 overexpressing mice. Seizures increase FGF-2 mRNA and protein levels in specific brain areas and upregulate the expression of its receptor FGFR-1. Short-term intrahippocampal injection of FGF-2 cause seizures, whereas long-term i.c.v. infusion of low-dose FGF-2 does not affect kainate seizures but promotes behavioral recovery and reduces hippocampal damage. Kainate seizure severity is not altered in FGF-2 knockout mice, but is increased in FGF-2 overexpressing mice. FGF-2 is implicated in seizure susceptibility and in seizure-induced plasticity.

Mechanical strain affects dura mater biological processes: implications for immature calvarial healing.

The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.

Expression of alternatively spliced FGF-2 antisense RNA transcripts in the central nervous system: regulation of FGF-2 mRNA translation

The fibroblast growth factor-2 (FGF-2) gene is bidirectionally transcribed to produce the FGF-2 mRNA and a 1.5 kb antisense (FGF-AS) transcript complementary to the 3′ untranslated region of the FGF-2 transcript. The FGF-AS RNA has been postulated to play a role in the post-transcriptional regulation of FGF-2, but this function has not been conclusively demonstrated. We characterized FGF-AS cDNAs from rat brain and C6 glioma cells, and investigated their role in regulation of FGF-2 expression. Three FGF-AS cDNAs were isolated; the full-length FGF-AS mRNA and two alternative splice variants lacking exon 2 or exons 2 and 3 of the FGF-AS sequence. The alternatively spliced FGF-AS RNAs are widely expressed in the CNS, whereas liver predominantly expressed the full-length transcript. The full-length and first splice variant encode 35 and 28 kDa isoforms of GFG, a MutT-related nuclear protein, whereas the second splice variant was not translated. The effect of FGF-AS RNA on FGF-2 expression was evaluated in stable C6 transfectants over-expressing the full-length or alternatively spliced FGF-AS RNA forms. All three constructs suppressed cellular FGF-2 protein (but not FGF-2 mRNA) levels, and this effect correlated directly with the level of FGF-AS RNA. Cellular FGF receptor content was increased and cell proliferation inhibited compared to wild type or vector-transfected cells, indicating disruption of the FGF-2 autocrine pathway by FGF-AS RNA. These findings demonstrate for the first time that the FGF-AS RNA regulates FGF-2 expression in mammalian cells, and suggest that this effect is exerted predominantly at the level of translation.

The nicotinic acetylcholine receptor agonist (±)-epibatidine increases FGF-2 mRNA and protein levels in the rat brain

In a previous work, we showed that acute intermittent nicotine treatment up-regulates the level of fibroblast growth factor-2 (FGF-2) mRNA in brain regions of tel- and mesencephalon of rats suggesting that neuroprotective effect of (−)nicotine may, at least in part, involve an activation of the neuronal FGF-2 signalling. The present experiments were designed to extend the study on the nicotinic receptor mediated up-regulation of FGF-2 mRNA levels to the use of the potent nicotinic acetylcholine receptor (nAChR) agonist (±)-epibatidine. The (±)-epibatidine treatment led to a strong and long lasting up-regulation of FGF-2 mRNA expression in the cerebral cortex, in the hippocampal formation, in the striatum and in the substantia nigra. This FGF-2 mRNA induction, already statistically significant at 4 h, peaked at 12 h from treatment and was only partially returned towards normal levels at 48 h, the last time point examined. Using Western blot analysis it was found that the epibatidine-induced upregulation of FGF-mRNA is accompaned by an increase of FGF-2 protein level at the 20-h time-interval. These (±)-epibatidine effects on FGF-2 expression were antagonized by the non-competitive nAChR antagonist mecamylamine, indicating an involvement of nicotinic receptors. In the same brain areas examined, no changes were observed in the fibroblast growth factor receptor-1 (FGFR-1) mRNA levels, in brain-derived neurotrophic factor (BDNF) and in glial cell line-derived neurotrophic factor (GDNF) mRNA levels. In view of the neurotrophic function of FGF-2, these results, together with previous ones, could further help to understand the molecular mechanisms mediating the previously observed neuroprotective effects of (−)nicotine.

Repression of Human Fibroblast Growth Factor 2 by a Novel Transcription Factor

Here we describe the cloning of the regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) using a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence. Overexpression of human RFT (RFT-A) reduces FGF-2 RNA and protein levels in both normal and tumor cell lines. Its splice variants, RFT-A' and RFT-B, have deletions in the putative DNA binding domain and fail to bind FGF-2 promoter and repress FGF-2 gene expression. The ratios of RFT isoforms differ between normal and tumor cells, with the splice variants dominating in tumor cells. Overexpression of RFT-A induces glioma cell death. Our data suggest that regulation of FGF-2 by RFT is important for cellular functions and may be impaired in certain tumors.

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.