C-type natriuretic peptide stimulates osteoblastic proliferation and collagen-X expression but suppresses fibroblast growth factor-23 expression in vitro.

BACKGROUND: The effects of C-type natriuretic peptide (CNP) and fibroblast growth factor (FGF)-23 appear to oppose each other during the process of bone formation, whereas few studies exist on the interaction between CNP and FGF-23. The main objective of the present study is to probe whether CNP is directly responsible for the regulation of osteoblast or via antagonizing FGF-23. METHODS: Osteoblasts were cultured in the absence or presence of CNP (0, 10, and 100 pmol/L) for 24 h, 48 h and 72 h, respectively. RESULTS: The findings of the present study indicated that: (1) CNP significantly stimulated osteoblastic proliferation and collagen (Col)-X expression; (2) both osteoblastic (osteocalcin, procollagen type I carboxy-terminal propeptide, total alkaline phosphatase and bone-specific alkaline phosphatase) and osteolytic (tartrate-resistant acid phosphatase and cross-linked carboxyterminal telopeptide of type I collagen) bone turnover biomarkers were up-regulated by CNP in osteoblasts; (3) FGF-23 mRNA and protein were significantly down-regulated at 24 h by CNP in osteoblasts, but the expression of FGF receptor-1/Klotho had no significant change. CONCLUSIONS: CNP stimulates osteoblastic proliferation and Col-X expression via the down-regulation of FGF-23 possibly in vitro. However, the specific mechanisms of the interaction between CNP and FGF-23 in osteoblasts are still unclear according to our findings. A further study on osteoblasts cultured with CNP and FGF-23 inhibitor will be undertaken in our laboratory.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo.

PURPOSE: To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. METHODS: Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. RESULTS: Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. CONCLUSIONS: The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

Up-regulation of fibroblast growth factor receptor 1 due to prenatal tobacco exposure can lead to developmental defects in new born.

Introduction: Tobacco-smoking is one of the most important risk factor for preterm delivery, pregnancy loss, low birth weight, and fetal growth restriction. It is estimated that approximately 30% of growth-restricted neonates could be independently associated with maternal smoking.Methods: In this study, gene expression profile, GSE11798, was chosen from GEO database with an aim to perceive change in gene expression signature in new born due to maternal smoking. Enrichment analysis was performed to annotate differentially expressed genes (DEGs) through gene ontology and pathway analysis using DAVID. Protein-protein interactions and module detection of these DEGs were carried out using cytoscape v3.6.0. Thirty umbilical cord tissue samples from 15 smokers and 15 non-smokers pregnant women were included in this analysis.Results: Twenty-six differentially expressed genes (DEGs) between two groups were selected using GEO2R tool. The DEGs were observed to be participating in biological processes/pathways related to growth releasing hormone, angiogenesis, embryonic skeletal, and cardiac development. Fibroblast growth factor receptor-1 (FGFR1) was identified to be the hub node with 348 interacting partners, which regulates transcription, cell growth, differentiation, and apoptosis. The up-regulation of FGFR1 in umbilical cord tissue may lead to reproductive and developmental complications such as encephalocraniocutaneous lipomatosis, osteoglophonic dysplasia, and Pfeiffer syndrome in new-borns.Conclusion: The findings manifests the possibility of overcoming these adverse health effects in new born through FGFR1 modulating treatments during pregnancy.

FGF21 promotes functional recovery after hypoxic-ischemic brain injury in neonatal rats by activating the PI3K/Akt signaling pathway via FGFR1/ß-klotho.

Perinatal asphyxia often results in neonatal cerebral hypoxia-ischemia (HI), which is associated with high mortality and severe long-term neurological deficits in newborns. Currently, there are no effective drugs to mitigate the functional impairments post-HI. Previous studies have shown that fibroblast growth factor 21 (FGF21) has a potential neuroprotective effect against brain injury. However, the effect of FGF21 on neonatal HI brain injury is unclear. In the present study, both in vivo and in vitro models were used to assess whether recombinant human FGF21 (rhFGF21) could exert a neuroprotective effect after HI and explore the associated mechanism. The results showed that the rhFGF21 treatment remarkably reduced the infarct volume, ameliorated the body weight and improved the tissue structure after HI in neonatal rats. In addition, the rhFGF21 treatment lengthened the running endurance times in the rotarod test and decreased the mean escape latencies and increased the number of platform crossings in the Morris water maze test at 21 d post-HI insult. In contrast, the FGFR1 inhibitor PD173074 and PI3K inhibitor LY294002 partially reversed these therapeutic effects. In isolated primary cortical neurons, the rhFGF21 treatment protected primary neurons from oxygen-glucose deprivation (OGD) insult by inhibiting neuronal apoptosis and promoting neuronal survival. Both our in vivo and in vitro results reveal that rhFGF21 could inhibit neuronal apoptosis by activating the PI3K/Akt signaling pathway via FGF21/FGFR1/ß-klotho complex formation. Therefore, rhFGF21 may be a promising therapeutic agent for promoting functional recovery after HI-induced neonatal brain injury.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo

Abstract Purpose To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. Methods Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. Results Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. Conclusions The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

Monoclonal antibody targeting of fibroblast growth factor receptor 1c causes cardiac valvulopathy in rats.

Fibroblast Growth Factors (FGFs) and their receptors (FGFRs) have been proposed as potential drug targets for the treatment of obesity. The aim of this study was to assess the potential toxicity in rats of three anti-FGFR1c mAbs with differential binding activity prior to clinical development. Groups of male rats received weekly injections of either one of two FGFR1c-specific mAbs or an FGFR1c/FGFR4-specific mAb at 10 mg/kg for up to 4 weeks. All three mAbs caused significant reductions in food intake and weight loss leading to some animals being euthanized early for welfare reasons. In all three groups given these mAbs, microscopic changes were seen in the bones and heart valves. In the bones of the femoro-tibial joint, thickening of the diaphyseal cortex of long bones, due to deposition of well organized new lamellar bone, indicated that an osteogenic effect was observed. In the heart, valvulopathy described as an endocardial myxomatous change affecting the mitral, pulmonary, tricuspid and aortic valves was observed in all mAb-treated animals. The presence of FGFR1 mRNA expression in the heart valves was confirmed using in situ hybridization. Targeting the FGF-FGFR1c pathway with anti-FGFR1c mAbs leads to drug induced valvulopathy in rats. In effect, this precluded the development of these mAbs as potential anti-obesity drugs. The valvulopathy observed was similar to that described for fenfluramine and dexafenfluramine. The pathogenesis of the drug-induced valvulopathy is considered FGFR1c-mediated, based on the specificity of the mAbs and FGFR1 mRNA expression in the heart valves.

Gabapentin regulates expression of FGF2 and FGFR1 in dorsal root ganglia via microRNA-15a in the arthritis rat model.

BACKGROUND: Arthritis is an inflammatory disease with a prevalence rate of approximately 10% in China, which commonly manifests as pain. The aim of the current study was to investigate the function of gabapentin in the dorsal root ganglion in an arthritis rat model, and assess the effect of gabapentin on the expression of fibroblast growth factor 2 (FGF2) and FGF receptor 1 (FGFR1). METHODS: A total of 30 healthy male Sprague-Dawley rats were randomly divided into the following three groups: Untreated group, control group and gabapentin group. Rats in the control and the gabapentin groups were injected with Freund's complete adjuvant to induce arthritis. A total of 7 days subsequent to model establishment, the gabapentin group was administered intraperitoneally gabapentin for 8 days. The alterations in thickness of paw pad and paw withdrawal mechanical threshold (PWMT) were detected, which indicated that the rats in the control and gabapentin groups presented with the symptoms of arthritis. RESULTS: In the control group, the PWMT value was significantly reduced (P < 0.05), whereas the PWMT value was significantly increased in the gabapentin group. Immunohistochemistry demonstrated that the expression levels of FGF2 and FGFR1 were increased in the control group compared with the untreated group, while the expression levels of FGF2 and FGFR1 were reduced in the gabapentin group. Moreover, the FGF2 antagonist PD173074 partially improved the plantar thickness and PWMT of the arthritic rats. Bioinformatics analysis predicted microRNA-15a binding sites in the 3'untranslated regions (UTR) of FGF2 and FGFR1. Furthermore, the expression of microRNA-15a was reduced in the control group compared with untreated rats, whereas microRNA-15a in the gabapentin group was upregulated compared with the control. Additionally, the luciferase reporter assay confirmed that microRNA-15a could inhibit the protein expression through pairing with the 3'UTR of FGF2 and FGFR1 mRNAs. CONCLUSION: Gabapentin may relieve arthritis pain and reduce the expression of FGF2 and FGFR1 in dorsal root ganglia. Furthermore, microRNA-15a may be involved in the regulatory process.

Green Tea Induced Cellular Proliferation and the Expression of Transforming Growth Factor-ß1 in the Jejunal Mucosa of Fasting Rats.

OBJECTIVE: The aim of this study was to understand whether or not the protective effect of green tea after fasting-induced damage in the jejunal mucosa of rat is dependent on cell proliferation and the stimulation of specific growth factors. MATERIALS AND METHODS: Sixty adult male Wistar rats were used in this study. The animals were divided randomly into 5 groups, with 12 in each group (G1-5). The animals in G1 (control group) were fed a rat chow diet and water ad libitum. The animals in G2 (fasting group) were fasted for 3 days. The animals in the G3, G4, and G5 groups were fasted for 3 days as G2, but were given water (G3), green tea (G4), or a vitamin E (G5) solution, respectively, for another 7 days. The animals were euthanized, and the jejunum was removed and processed for histological and immunohistochemical analysis. RESULTS: Compared to the G3 group, the jejunal mucosa of G4 rats showed a 70.6% higher level (p < 0.001) of expression of proliferating cell nuclear antigen and 98% higher level (p = 0.0001) of the expression of transforming growth factor-ß1 (TGF-ß1), whereas the level of fibroblast growth factor-1 (FGF-1) and insulin-like growth factor-1 (IGF-1) expression was 22 and 11% lower, respectively, in G4 animals as compared to G3 rats. These differences in the expression of FGF-1 and IGF-1 in G4 animals were not statistically significant. CONCLUSION: In this study, green tea repaired the fasting-induced damage in the jejunal mucosa of rats, mainly by inducing a significant expression of TGF-ß1 in the jejunal mucosa.

FGF2 Prevents Sunitinib-Induced Cardiotoxicity in Zebrafish and Cardiomyoblast H9c2 Cells.

Sunitinib is used extensively in the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. However, the undesirable cardiotoxic effects of sunitinib, such as congestive heart failure and hypertension, limit its use in the clinical setting. As multiple receptor tyrosine kinases are inhibited by sunitinib, it raises a question as to which target mediates sunitinib-induced cardiotoxicity. Here, we reported that the injection of fibroblast growth factor 2 (FGF2) mRNA into one- to two-cell stage embryos protected against sunitinib-induced cardiotoxicity in zebrafish. In addition, FGF2 significantly prevented sunitinib-induced cardiotoxicity in cardiomyoblast H9c2 cells, possibly via activating the PLC-γ/c-Raf/CREB pathway. Importantly, FGF2 did not compromise the antitumor activity of sunitinib in Caki-1 and OS-RC-2 renal cell carcinoma cells. Molecular docking simulations further revealed an interaction between the tyrosine kinase domain of FGF receptor 1 (FGFR1) and sunitinib. Taken together, our results clearly demonstrated that FGF2 inhibition plays an important role in sunitinib-induced cardiotoxicity both in vitro and in vivo. This study also provided a basis for further research on sunitinib-induced cardiotoxicity and may allow rational design of new sunitinib derivatives with fewer or weak cardiotoxic effects.

Sequential exposure to fibroblast growth factors (FGF) 2, 9 and 18 enhances hMSC chondrogenic differentiation.

OBJECTIVE: To test the effects of sequential exposure to FGF2, 9 and 18 on human Mesenchymal Stem Cells (hMSC) differentiation during in vitro chondrogenesis. DESIGN: Control and FGF2-expanded hMSC were cultured in aggregates in the presence of rhFGF9, rhFGF18 or rhFGFR3-specific signaling FGF variants, starting at different times during the chondroinductive program. Quantitative real time polymerase chain reaction (qRT-PCR) and immunocytochemistry were performed at different stages. The aggregate cultures were switched to a hypertrophy-inducing medium along with rhFGFs and neutralizing antibodies against FGFR1 and FGFR3. Histological/immunohistochemical/biochemical analyses were performed. RESULTS: FGF2-exposed hMSC during expansion up-regulated Sox9 suggesting an early activation of the chondrogenic machinery. FGF2, FGF9 and 18 modulated the expression profile of FGFR1 and FGFR3 in hMSC during expansion and chondrogenesis. In combination with transforming growth factor-beta (TGF-ß), FGF9 and FGF18 inhibited chondrogenesis when added at the beginning of the program (≤ d7), while exhibiting an anabolic effect when added later (≥d14), an effect mediated by FGFR3. Finally, FGFR3 signaling induced by either FGF9 or FGF18 delayed the appearance of spontaneous and induced hypertrophy-related changes. CONCLUSIONS: The stage of hMSC-dependent chondrogenesis at which the growth factors are added impacts the progression of the differentiation program: increased cell proliferation and priming (FGF2); stimulated early chondrogenic differentiation (TGF-ß, FGF9/FGF18) by shifting the chondrogenic program earlier; augmented extracellular matrix (ECM) production (FGF9/FGF18); and delayed terminal hypertrophy (FGF9/FGF18). Collectively, these factors could be used to optimize pre-implantation conditions of hMSC when used to engineer cartilage grafts.

MicroRNA-34a negatively regulates anesthesia-induced hippocampal apoptosis and memory impairment through FGFR1.

BACKGROUND: Mounting evidence has shown the toxic effects of anesthesia to neonatal hippocampus. We used an in vivo mouse model to explore the role of microRNA 34a (miR-34a) in regulating anesthesia-induced hippocampal neurotoxicity. METHODS: One-month old C57/BL6 mice received daily intraperitoneal injection of anesthesia (ketamine, 50 mg/kg) for 7 days. One day after, apoptosis was evaluated by TUNEL staining in hippocampal CA1 region, and expression level of miR-34a assessed by real-time quantitative PCR (qPCR). Hippocampal miR-34a was then down-regulated through lentivirus mediated cortical injection prior to anesthesia. The effects of inhibiting hippocampal miR-34a on anesthesia-induced hippocampal apoptosis and memory impairment were further investigated by TUNEL staining and Morris water maze (MWM) test. The predicted molecular target of miR-34a, fibroblast growth factor receptor 1 (FGFR1) was down-regulated in hippocampus through siRNA-mediated cortical injection and its effect on hippocampal apoptosis was also examined. RESULTS: Anesthesia caused severe apoptosis among hippocampal CA1 neurons and upregulated hippocampal miR-34a. On the other hand, lentivirual inhibition of miR-34a protected anesthesia-induced hippocampal apoptosis and memory impairment. Luciferase essay demonstrated FGFR1 was directly regulated by miR-34a in hippocampus. siRNA-induced FGFR1 downregulation further exaggerated anesthesia-induced apoptosis in hippocampus. CONCLUSIONS: Overall, we showed that miR-34a negatively modulated anesthesia-induced hippocampal neurotoxicity.

Fibroblast growth factor receptor inhibition is active against mesothelioma and synergizes with radio- and chemotherapy.

RATIONALE: Malignant pleural mesothelioma is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy, poor outcome, and limited therapeutic options. Fibroblast growth factors (FGFs) and their receptors are potential targets for cancer therapy, but their significance in mesothelioma has remained largely undefined. OBJECTIVES: To investigate the antimesothelioma potential of FGF receptor 1 (FGFR1) inhibition. METHODS: Expression of FGFs and their receptors was analyzed in mesothelioma cell lines and tissue specimens. Several cell models were used to investigate FGFR1 inhibition in vitro and in combination with cisplatin and irradiation. Mouse intraperitoneal xenotransplant models were used for in vivo validation. MEASUREMENTS AND MAIN RESULTS: FGFR1, FGF2, and FGF18 were overexpressed in mesothelioma. Stimulation with FGF2 led to increased cell proliferation, migration, and transition to a more sarcomatoid phenotype in subsets of mesothelioma cell lines. In contrast, inhibition of FGFR1 by a specific kinase inhibitor or a dominant-negative FGFR1 construct led to significantly decreased proliferation, clonogenicity, migration, spheroid formation, and G1 cell cycle arrest in several mesothelioma cell lines, accompanied by apoptosis induction and decreased mitogen-activated protein kinase pathway activity. Reduced tumor growth, proliferation, mitogenic signaling, and apoptosis induction were observed in vivo. Inhibition of FGFR1 synergistically enhanced the cytotoxic effects of ionizing radiation and cisplatin. CONCLUSIONS: Our data suggest that the malignant phenotype of mesothelioma cells depends on intact FGF signals, which should be considered as therapeutic targets with a promising chemo- and radiosensitizing potential.

Effect of heparin-derived oligosaccharide on bFGFR1 and bFGFR2 in vascular smooth muscle cells.

Our purpose is to investigate the inhibitory effect and mechanisms of heparin-derived oligosaccharide (HDO) on proliferation of vascular smooth muscle cells (VSMCs) induced by basic fibroblast growth factor (bFGF). Proliferation of VSMCs was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; cell cycle distribution was analyzed by flow cytometry; bFGF receptor 1 and receptor 2 (bFGFR1 and bFGFR2) messenger RNA (mRNA) expression levels were determined by reverse transcription-polymerase chain reaction; and its protein expression levels were detected by Western blotting and immunocytochemical methods. Results showed that HDO inhibited VSMC proliferation in a dose-dependent manner; HDO inhibited cells in G1 phase entering the S phase; HDO inhibited bFGFR1 and bFGFR2 mRNA expression levels. In addition, bFGFR1 and bFGFR2 protein expression levels were significantly inhibited by HDO dose dependently. These results imply that HDO can inhibit VSMC proliferation. The proliferation of bFGF-induced VSMCs by HDO is associated with the inhibition of bFGFR1 and bFGFR2 expression levels. This altered molecular signature may explain one mechanism of HDO-mediated inhibition of VSMC proliferation.

Paricalcitol downregulates myocardial renin-angiotensin and fibroblast growth factor expression and attenuates cardiac hypertrophy in uremic rats.

BACKGROUND: Vitamin D attenuates uremic cardiac hypertrophy, possibly by suppressing the myocardial renin-angiotensin system (RAS) and fibroblast growth factors (FGFs). We compared the suppression of cardiac hypertrophy and myocardial expression of RAS and FGF receptor genes offered by the vitamin D analog paricalcitol (Pc) or the angiotensin-converting enzyme inhibitor enalapril (E) in experimental uremia. METHODS: Rats with 5/6 nephrectomy received Pc or E for 8 weeks. Renal function, systolic blood pressure, and cardiac hypertrophy were evaluated. Myocardial expression of RAS genes, brain natriuretic peptide (BNP), and FGF receptor-1 (FGFR-1) were determined using quantitative reverse-transcription (pRT)-PCR. RESULTS: Blood pressure, proteinuria, and serum creatinine were significantly higher in untreated uremic animals. Hypertension was significantly reduced by E but only modestly by Pc; however, cardiac hypertrophy in the untreated group was similarly attenuated by Pc or E. Upregulation of myocardial expressions of renin, angiotensinogen, FGFR-1, and BNP in untreated uremic animals was reduced similarly by Pc and E, while the angiotensin II type 1 receptor was downregulated only by E. CONCLUSIONS: Uremic cardiac hypertrophy is associated with activation of the myocardial RAS and the FGFR-1. Downregulation of these genes induced by Pc and E results in similar amelioration of left ventricular hypertrophy despite the different antihypertensive effects of these drugs.

Pien Tze Huang inhibits tumor angiogenesis in a mouse model of colorectal cancer via suppression of multiple cellular pathways.

Angiogenesis plays an essential role in cancer progression, which therefore has become an attractive target for anticancer treatment. Tumor angiogenesis is tightly regulated by multiple signaling pathways that usually function redundantly; in addition, crosstalk between these pathways forms a complicated network that is regulated by compensatory mechanisms. Given the complexity of pathogenic mechanisms underlying tumor angiogenesis, most currently used angiogenesis inhibitors that only target single pathways may be insufficient and probably generate drug resistance, thus, increasing the necessity for development of novel anticancer agents. Traditional Chinese medicines (TCM) are receiving great interest since they have relatively fewer side-effects and have been used for thousands of years to clinically treat various types of diseases including cancer. Pien Tze Huang (PZH), a well-known traditional Chinese formulation that was first prescribed 450 years ago, has long been used as an alternative remedy for cancers. However, the precise mechanism of PZH's anticancer activity remains to be further elucidated. Using a colorectal cancer mouse xenograft model, in the present study, we evaluated the effect of PZH on tumor angiogenesis and investigated the underlying molecular mechanisms. We found that PZH inhibited tumor growth since PZH treatment resulted in decrease in both tumor volume and tumor weight in CRC mice. In addition, PZH suppressed the activation of several signaling pathways such as STAT3, Akt and MAPKs. Consequently, the inhibitory effect of PZH on these pathways resulted in the inhibition of tumor angiogenesis as demonstrated by the decrease of microvessel density in tumor tissues. Moreover, PZH treatment reduced the expression of angiogenic factors including iNOS, eNOS, VEGF-A, bFGF as well as their specific receptors VEGFR2 and bFGFR. Altogether, our findings suggest that inhibition of tumor angiogenesis via suppression of multiple signaling pathways might be one of the mechanisms whereby PZH affects cancers.

SUN11602, a novel aniline compound, mimics the neuroprotective mechanisms of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) offers some measure of protection against excitotoxic neuronal injuries by upregulating the expression of the calcium-binding protein calbindin-D28k (Calb). The newly synthesized small molecule 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) mimics the neuroprotective effects of bFGF, and thus, we examined how SUN11602 exerts its actions on neurons in toxic conditions of glutamate. In primary cultures of rat cerebrocortical neurons, SUN11602 and bFGF prevented glutamate-induced neuronal death. This neuroprotection, which occurred in association with the augmented phosphorylation of the bFGF receptor-1 (FGFR-1) and the extracellular signal-regulated kinase-1/2 (ERK-1/2), was abolished by pretreatment with PD166866 (a FGFR-1 tyrosine kinase-specific inhibitor) and PD98059 (a mitogen-activated protein kinase [MAPK]/[ERK-1/2] kinase [MEK] inhibitor). In addition, SUN11602 and bFGF increased the levels of CALB1 gene expression in cerebrocortical neurons. Whether this neuroprotection was linked to Calb was investigated with primary cultures of cerebrocortical neurons from homozygous knockout (Calb(-/-)) and wild-type (WT) mice. In WT mice, SUN11602 and bFGF increased the levels of newly synthesized Calb in cerebrocortical neurons and suppressed the glutamate-induced rise in intracellular Ca(2+). This Ca(2+)-capturing ability of Calb allowed the neurons to survive severe toxic conditions of glutamate. In contrast, Calb levels remained unchanged in Calb(-/-) mice after exposure to SUN11602 or bFGF, and due to a loss of function of the gene, these neurons were no longer resistant to toxic conditions of glutamate. These findings indicated that SUN11602 activated a number of cellular molecules (FGFR-1, MEK/ERK intermediates, and Calb) and consequently contributed to intracellular Ca(2+) homeostasis as observed in the case of bFGF.

Ginsenoside-Rg1 induces angiogenesis via non-genomic crosstalk of glucocorticoid receptor and fibroblast growth factor receptor-1.

AIMS: Ginsenoside-Rg1, the most prevalent active constituent of Panax ginseng, has been shown to possess potent pro-angiogenic properties and therefore poses special interest for the development as a novel modality for angiotherapy. Rg1 can activate the glucocorticoid receptor (GR). However, the mechanism that transmits these pro-angiogenic effects is still unclear. METHODS AND RESULTS: By using human umbilical vein endothelial cells (HUVECs), we show for the first time that in the presence of Rg1, GR and fibroblast growth factor receptor-1 (FGFR-1) cooperate to activate a non-genomic signalling cascade that results in angiogenic activity. The activation of FGFR-1 by Rg1 was blocked by the GR antagonist RU486. Depletion of FGFR-1 expression or inhibition of its activity using small interfering RNA and small molecule inhibitor, respectively, significantly inhibited Rg1-induced phosphatidylinositol 3-kinase/Akt phosphorylation and subsequent endothelial nitric oxide synthase activation and angiogenic tube formation, confirming that the effect was FGFR-1 specific. On exploring how GR might regulate the activation of FGFR-1, we found that GR-mediated FGFR-1 activation was ligand-independent. In addition, we have shown that FGFR-1 regulation by GR was associated with GR/FGFR-1 complex formation. CONCLUSION: This study provides important new insights into the mechanism regarding the beneficial effects of Rg1 on angiogenesis. We propose that Rg1 could be a novel prototype of nutraceutical that can induce therapeutic angiogenesis.

Perturbation of Fgf10 signal pathway in mouse embryonic palate by dexamethasone and vitamin B12 in vivo.

BACKGROUND/PURPOSE: The Fgf10 signaling pathway plays an important role in early stages of mouse embryonic palatal development, which is associated with cell proliferation and differentiation. The objective of this study was to assess whether dexamethasone and vitamin B(12) affected the Fgf10 signal pathway of mouse embryonic palate. MATERIALS AND METHODS: Immunohistochemical studies were performed for expression of Fgf10, Fgfr2b, and sonic hedgehog and for cell proliferation and apoptosis of mouse embryonic palate. RESULTS: The expression of Fgf10, Fgfr2b, and sonic hedgehog was changed in mouse embryonic palate after dexamethasone and vitamin B(12) treatment, resulting in reduced and restored proliferation of mesenchymal cells. CONCLUSIONS: Dexamethasone and vitamin B(12) affected the Fgf10 signaling pathway and cell proliferation of mouse embryonic palate. Cell apoptosis was not altered after dexamethasone and vitamin B(12) exposure.

NCAM-mimetic, FGL peptide, restores disrupted fibroblast growth factor receptor (FGFR) phosphorylation and FGFR mediated signaling in neural cell adhesion molecule (NCAM)-deficient mice.

Neural cell adhesion molecule (NCAM) is a membrane-bound glycoprotein expressed on the surface of neuronal and glial cells. Previous in vitro studies have demonstrated that NCAM promotes neuronal functions largely via three main interaction partners: the fibroblast growth factor receptor (FGFR), a member of Src family of tyrosine kinases, Fyn and Raf1 kinase which all activate different intracellular signaling pathways. The objective was to clarify, which signaling pathways are being disrupted in NCAM knockout mice and whether FGL peptide is able to restore observed disruptions. Therefore we compared the levels of phosphorylation of FGFR1, Src kinase Fyn, Raf1 kinase, MAP kinases, Akt kinase and calcium/calmodulin-dependent kinases II and IV (CaMKII and CaMKIV) in the hippocampus of NCAM knockout mice to their wild-type littermates. The data of our study show that mice constitutively deficient in all isoforms of NCAM have decreased basal phosphorylation levels of FGFR1 and CaMKII and CaMKIV. Furthermore, NCAM-mimetic, FGL peptide, is found to be able to restore FGFR1, CaMKII and CaMKIV phosphorylation levels and thereby mimic the interactions of NCAM at this receptor in NCAM deficient mice. Also, we found that Fyn(Tyr530), Raf1, MAP kinases and Akt kinase phosphorylation in adult animals is not affected by NCAM deficiency but interestingly, we found an over-expression of another cell adhesion molecule L1. We conclude that in NCAM deficient mice FGFR1-dependent signaling is disrupted and it can be restored by FGL peptide.

Specificity of the second messenger pathways involved in basic fibroblast growth factor-induced survival and neurite growth in chick ciliary ganglion neurons.

Basic fibroblast growth factor (bFGF) exerts multiple neurotrophic actions on cultured neurons from the ciliary ganglion of chick embryo, among them promotion of neuronal survival and of neurite outgrowth. To understand the specificity of the signal transduction cascades involved in the control of these processes, we used pharmacological inhibitors of the three main effectors known to act downstream of the bFGF receptor (FGFR): phospholipase Cgamma (PLCgamma), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Neuronal survival was assessed at 24 and 48 hr; neurite growth was analyzed both on dissociated neurons and on explants of whole ganglia. Our data show that only the PI3-K pathway is involved in the survival-promoting effect of bFGF; on the other hand, all three effectors converge on the enhancement of neurite outgrowth, both on isolated neurons and in whole ganglia.