RESUMEN
In recent years, a surprisingly complex picture emerged about endoplasmic reticulum (ER)/Golgi-independent secretory pathways, and several routes have been discovered that differ with regard to their molecular mechanisms and machineries. Fibroblast growth factor 2 (FGF2) is secreted by a pathway of unconventional protein secretion (UPS) that is based on direct self-translocation across the plasma membrane. Building on previous research, a component of this process has been identified to be glypican-1 (GPC1), a GPI-anchored heparan sulfate proteoglycan located on cell surfaces. These findings not only shed light on the molecular mechanism underlying this process but also reveal an intimate relationship between FGF2 and GPC1 that might be of critical relevance for the prominent roles they both have in tumor progression and metastasis.
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Factor 2 de Crecimiento de Fibroblastos , Aparato de Golgi , Animales , Transporte Biológico , Membrana Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Aparato de Golgi/metabolismo , Mamíferos , Transporte de ProteínasRESUMEN
Phosphoinositides are amphipathic lipid molecules derived from phosphatidylinositol that represent low abundance components of biological membranes. Rather than serving as mere structural elements of lipid bilayers, they represent molecular switches for a broad range of biological processes, including cell signaling, membrane dynamics and remodeling, and many other functions. Here, we focus on the molecular mechanisms that turn phosphoinositides into molecular switches and how the dysregulation of these processes can lead to disease.
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Enfermedad , Fosfatidilinositoles , Transducción de Señal , Membrana Celular/metabolismo , Fosfatidilinositoles/metabolismo , HumanosRESUMEN
The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24â h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.
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Nucléolo Celular , Factor 2 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Nucléolo Celular/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Transcripción Genética , ADN Ribosómico/genética , Cromatina/genética , Cromatina/metabolismoRESUMEN
Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFß1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFß1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.
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Factor 2 de Crecimiento de Fibroblastos , Organoides , Adulto , Diferenciación Celular/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Glándulas Salivales , Análisis de Secuencia de ARN , Células del Estroma/metabolismoRESUMEN
Fibroblast growth factor-2 (FGF2) is involved in the regulation of affective behaviour and shows antidepressant effects through the Akt and extracellular signal regulated kinase (ERK) 1/2 pathways. Nudix hydrolase 6 (NUDT6) protein is encoded from FGF2 gene's antisense strand and its role in the regulation of affective behaviour is unknown. Here, we overexpressed NUDT6 in the hippocampus and investigated its behavioural effects and the underlying molecular mechanisms affecting the behaviour. We showed that increasing hippocampal NUDT6 results in depression-like behaviour in rats without changing FGF2 levels or activating its downstream effectors, Akt and ERK1/2. Instead, NUDT6 acted by inducing inflammatory signalling, specifically by increasing S100 calcium binding protein A9 (S100A9) levels, activating nuclear factor-kappa B-p65 (NF-κB-p65), and elevating microglia numbers along with a reduction in neurogenesis. Our results suggest that NUDT6 could play a role in major depression by inducing a proinflammatory state. This is the first report of an antisense protein acting through a different mechanism of action than regulation of its sense protein. The opposite effects of NUDT6 and FGF2 on depression-like behaviour may serve as a mechanism to fine-tune affective behaviour. Our findings open up new venues for studying the differential regulation and functional interactions of sense and antisense proteins in neural function and behaviour, as well as in neuropsychiatric disorders. KEY POINTS: Hippocampal overexpression of nudix hydrolase 6 (NUDT6), the antisense protein of fibroblast growth factor-2 (FGF2), increases depression-like behaviour in rats. Hippocampal NUDT6 overexpression triggers a neuroinflammatory cascade by increasing S100 calcium binding proteinA9 (S100A9) expression and nuclear NF-κB-p65 translocation in neurons, in addition to microglial recruitment and activation. Hippocampal NUDT6 overexpression suppresses neurogenesis. NUDT6 exerts its actions without altering the levels or downstream signalling pathways of FGF2.
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Depresión , Factor 2 de Crecimiento de Fibroblastos , FN-kappa B , Animales , Ratas , Factor 2 de Crecimiento de Fibroblastos/genética , Inflamación/genética , Neurogénesis/genética , FN-kappa B/metabolismo , FN-kappa B/farmacología , Proteínas Proto-Oncogénicas c-akt , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Depresión/genética , Depresión/metabolismoRESUMEN
Fibroblast growth factors (FGFs) control various cellular functions through fibroblast growth factor receptor (FGFR) activation, including proliferation, differentiation, migration, and survival. FGFR amplification in ER + breast cancer patients correlate with poor prognosis, and FGFR inhibitors are currently being tested in clinical trials. By comparing three-dimensional spheroid growth of ER + breast cancer cells with and without FGFR1 amplification, our research discovered that FGF2 treatment can paradoxically decrease proliferation in cells with FGFR1 amplification or overexpression. In contrast, FGF2 treatment in cells without FGFR1 amplification promotes classical FGFR proliferative signaling through the MAPK cascade. The growth inhibitory effect of FGF2 in FGFR1 amplified cells aligned with an increase in p21, a cell cycle inhibitor that hinders the G1 to S phase transition in the cell cycle. Additionally, FGF2 addition in FGFR1 amplified cells activated JAK-STAT signaling and promoted a stem cell-like state. FGF2-induced paradoxical effects were reversed by inhibiting p21 or the JAK-STAT pathway and with pan-FGFR inhibitors. Analysis of patient ER + breast tumor transcriptomes from the TCGA and METABRIC datasets demonstrated a strong positive association between expression of FGF2 and stemness signatures, which was further enhanced in tumors with high FGFR1 expression. Overall, our findings reveal a divergence in FGFR signaling, transitioning from a proliferative to stemness state driven by activation of JAK-STAT signaling and modulation of p21 levels. Activation of these divergent signaling pathways in FGFR amplified cancer cells and paradoxical growth effects highlight a challenge in the use of FGFR inhibitors in cancer treatment.
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Neoplasias de la Mama , Transducción de Señal , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Quinasas Janus/metabolismo , Quinasas Janus/farmacología , Quinasas Janus/uso terapéutico , Factores de Transcripción STAT/metabolismo , Factores de Transcripción STAT/farmacología , Factores de Transcripción STAT/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Proliferación Celular , Factores de Crecimiento de Fibroblastos/farmacología , Línea Celular TumoralRESUMEN
OBJECTIVE: Anesthetics have been linked to cognitive alterations, particularly in the elderly. The current research delineates how Fibroblast Growth Factor 2 (Fgf2) modulates tau protein phosphorylation, contributing to cognitive impairments in aged rats upon sevoflurane administration. METHODS: Rats aged 3, 12, and 18 months were subjected to a 2.5% sevoflurane exposure to form a neurotoxicity model. Cognitive performance was gauged, and the GEO database was employed to identify differentially expressed genes (DEGs) in the 18-month-old cohort post sevoflurane exposure. Bioinformatics tools, inclusive of STRING and GeneCards, facilitated detailed analysis. Experimental validations, both in vivo and in vitro, examined Fgf2's effect on tau phosphorylation. RESULTS: Sevoflurane notably altered cognitive behavior in older rats. Out of 128 DEGs discerned, Fgf2 stood out as instrumental in regulating tau protein phosphorylation. Sevoflurane exposure spiked Fgf2 expression in cortical neurons, intensifying tau phosphorylation via the PI3K/AKT/Gsk3b trajectory. Diminishing Fgf2 expression correspondingly curtailed tau phosphorylation, neurofibrillary tangles, and enhanced cognitive capacities in aged rats. CONCLUSION: Sevoflurane elicits a surge in Fgf2 expression in aging rats, directing tau protein phosphorylation through the PI3K/AKT/Gsk3b route, instigating cognitive aberrations.
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Anestésicos por Inhalación , Disfunción Cognitiva , Éteres Metílicos , Anciano , Animales , Humanos , Lactante , Ratas , Anestésicos por Inhalación/efectos adversos , Anestésicos por Inhalación/metabolismo , Cognición , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/metabolismo , Hipocampo/metabolismo , Éteres Metílicos/farmacología , Éteres Metílicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sevoflurano/metabolismo , Sevoflurano/farmacología , Proteínas tau/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismoRESUMEN
Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.
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Factor 2 de Crecimiento de Fibroblastos , Técnicas de Maduración In Vitro de los Oocitos , Animales , Ratones , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factores de Crecimiento Endotelial/análisis , Factores de Crecimiento Endotelial/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Oocitos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Suplementos Dietéticos , Glucosa/farmacología , Glucosa/metabolismo , Células del Cúmulo/metabolismoRESUMEN
Despite various clinical options, human anterior cruciate ligament (ACL) lesions do not fully heal. Biomaterial-guided gene therapy using recombinant adeno-associated virus (rAAV) vectors may improve the intrinsic mechanisms of ACL repair. Here, we examined whether poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can deliver rAAV vectors coding for the reparative basic fibroblast growth factor (FGF-2) and transforming growth factor beta (TGF-ß) in human mesenchymal stromal cells (hMSCs) as a source of implantable cells in ACL lesions. Efficient and sustained rAAV-mediated reporter (red fluorescent protein) and therapeutic (FGF-2 and TGF-ß) gene overexpression was achieved in the cells for at least 21 days in particular with pNaSS-grafted PCL films relative to all other conditions (up to 5.2-fold difference). Expression of FGF-2 and TGF-ß mediated by rAAV using PCL films increased the levels of cell proliferation, the DNA contents, and the deposition of proteoglycans and of type-I and -III collagen (up to 2.9-fold difference) over time in the cells with higher levels of transcription factor expression (Mohawk, Scleraxis) (up to 1.9-fold difference), without activation of inflammatory tumor necrosis alpha especially when using pNaSS-grafted PCL films compared with the controls. Overall, the effects mediated by TGF-ß were higher than those promoted by FGF-2, possibly due to higher levels of gene expression achieved upon rAAV gene transfer. This study shows the potential of using functionalized PCL films to apply rAAV vectors for ACL repair.
RESUMEN
INTRODUCTION/AIMS: Daily intramuscular injections of fibroblast growth factor 2 (FGF2) but not of brain-derived neurotrophic factor (BDNF) significantly improve whisking behavior and mono-innervation of the rat levator labii superioris (LLS) muscle 56 days after buccal nerve transection and suture (buccal-buccal anastomosis, BBA). We explored the dose-response of BDNF, FGF2, and insulin growth factor 2 (IGF2) on the same parameters, asking whether higher doses of BDNF would promote recovery. METHODS: After BBA, growth factors were injected (30 µL volume) daily into the LLS muscle over 14, 28, or 56 days. At 56 days, video-based motion analysis of vibrissal whisking was performed and the extent of mono- and poly-reinnervation of the reinnervated neuromuscular junctions (NMJs) of the muscle determined with immunostaining of the nerve with ß-tubulin and histochemical staining of the endplates with Alexa Fluor 488-conjugated α-bungarotoxin. RESULTS: The dose-response curve demonstrated significantly higher whisking amplitudes and corresponding increased mono-innervation of the NMJ in the reinnervated LLS muscle at concentrations of 20-30 µg/mL BDNF administered daily for 14-28 days after BBA surgery. In contrast, high doses of IGF2 and FGF2, or doses of 20 and 40 µg/mL of BDNF administered for 14-56 days had no effect on either whisking behavior or in reducing poly-reinnervation of endplates in the muscle. DISCUSSION: These data suggest that the re-establishment of mono-innervation of whiskerpad muscles and the improved motor function by injections of BDNF into the paralyzed vibrissal musculature after facial nerve injury have translation potential and promote clinical application.
Asunto(s)
Traumatismos del Nervio Facial , Ratas , Animales , Traumatismos del Nervio Facial/tratamiento farmacológico , Factor Neurotrófico Derivado del Encéfalo/farmacología , Inyecciones Intramusculares , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Unión Neuromuscular , Regeneración Nerviosa/fisiología , Recuperación de la Función/fisiología , Nervio FacialRESUMEN
Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2-dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.
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Adipogénesis , Tejido Adiposo/patología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , MicroARNs/genética , Músculo Esquelético/patología , Osteonectina/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Tejido Adiposo/metabolismo , Anciano , Diferenciación Celular , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Músculo Esquelético/metabolismo , Osteonectina/genética , Proteínas Proto-Oncogénicas c-fos/genética , Transducción de SeñalRESUMEN
Retinal neovascularization is a leading cause of severe visual loss in humans, and molecular mechanisms of microglial activation-driven angiogenesis remain unknown. Using single-cell RNA sequencing, we identified a subpopulation of microglia named sMG2, which highly expressed necroptosis-related genes Rip3 and Mlkl. Genetic and pharmacological loss of function demonstrated that hypoxia-induced microglial activation committed to necroptosis through the RIP1/RIP3-mediated pathway. Specific deletion of Rip3 gene in microglia markedly decreased retinal neovascularization. Furthermore, hypoxia induced explosive release of abundant FGF2 in microglia through RIP3-mediated necroptosis. Importantly, blocking signaling components of the microglia necropotosis-FGF2 axis largely ablated retinal angiogenesis and combination therapy with simultaneously blocking VEGF produced synergistic antiangiogenic effects. Together, our data demonstrate that targeting the microglia necroptosis axis is an antiangiogenesis therapy for retinal neovascular diseases.
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Microglía/patología , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Enfermedades de la Retina/patología , Animales , Quimioterapia Combinada , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Hipoxia/patología , Ratones , Microglía/metabolismo , Necroptosis/efectos de los fármacos , Neovascularización Patológica , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/metabolismo , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Acute lymphoblastic leukemia (ALL) and glioma are some of the most common malignancies, with ALL most often affecting children and glioma affecting adult men. Proangiogenic cytokines and growth factors play an important role in the development of both of these tumors. Glioma is characterized by an extremely extensive network of blood vessels, which continues to expand mainly in the process of neoangiogenesis, the direct inducers of which are cytokines from the family of vascular endothelial growth factors, i.e., vascular endothelial growth factor (VEGF-A) and its receptor vascular endothelial growth factor receptor 2 (VEGF-R2), as well as a cytokine from the fibroblast growth factor family, fibroblast growth factor 2 (FGF-2 or bFGF). Growth factors are known primarily for their involvement in the progression and development of solid tumors, but there is evidence that local bone marrow angiogenesis and increased blood vessel density are also present in hematological malignancies, including leukemias. The aim of this study was to examine changes in the concentrations of VEGF-A, VEGF-R2, and FGF-2 (with a molecular weight of 17 kDa) in a group of patients divided into specific grades of malignancy (glioma) and a control group; changes of VEGF-A and FGF-2 concentrations in childhood acute lymphoblastic leukemia and a control group; and to determine correlations between the individual proteins as well as the influence of the patient's age, diet, and other conditions that may place the patient in the risk group. During the statistical analysis, significant differences in concentrations were found between the patient and control groups in samples from people with diagnosed glioma and from children with acute lymphoblastic leukemia, but in general, there are no significant differences in the concentrations of VEGF-A, VEGF-R2, and FGF-2 between different grades of glioma malignancy. Among individuals treated for glioma, there was no significant impact from the patient's gender and age, consumption of food from plastic packaging, frequency of eating vegetables and fruit, smoking of tobacco products, the intensity of physical exercise, or the general condition of the body (Karnofsky score) on the concentrations of the determined cytokines and receptor. The listed factors do not bring about an actual increase in the risk of developing brain glioma.
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Glioma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Masculino , Adulto , Niño , Humanos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Citocinas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Glioma/metabolismo , Encéfalo/metabolismoRESUMEN
Some hypotheses include schizophrenia as a neurodevelopmental disorder, which indicates a special role in growth factors and neuroglia in the development of schizophrenia symptoms. Growth factors are cytokine molecules that play an important role in the regulation of tissue nucleation, cell development, survival, and migration of all tissues in organisms, including the brain and nervous system. The aim of the study was to determine the serum concentration of six growth factors (EGF, VEGF, FGF-2, TGF-α, PDGF-AA, PDGF-AB/BB) in schizophrenia patients and to identify the correlations with clinical characteristics. After signing an informed consent form, 236 schizophrenia patients (F20 according to the ICD-10) and 102 healthy people were recruited in the study. In patients with schizophrenia, we observed a significant elevation in the TGF-α and PDGF-AA serum levels. The duration of schizophrenia was significantly positively correlated with the FGF-2 level. The PANSS total score had a positive correlation with the FGF-2 level and a negative correlation with the TGF-α level. Our results and literature indicate the involvement of growth factors in the mechanisms of development of schizophrenia. Combined biomarker screening seems to be necessary to improve diagnosis and clinical follow-up of patients with severe mental illnesses.
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Mechanical stimuli serve as crucial regulators of bone mass, promoting bone formation. However, the molecular mechanisms governing how mesenchymal stem cells (MSCs) respond to mechanical cues during their differentiation into osteogenic cells remain elusive. In this study, we found that cyclic stretching enhances MSC proliferation but does not increase the expression of osteoblast-related genes. We further revealed that this proliferative effect is mediated by fibroblast growth factor 2 (FGF-2), synthesized by MSCs in response to mechanical stress. Cell proliferation induced by cyclic stretching was inhibited upon the addition of either U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), or early growth response 1 (EGR1)-targeting small-hairpin RNA (shRNA), indicating the involvement of the extracellular signal-regulated kinase (ERK)/EGR1 signaling pathway. Osteoblast differentiation, evaluated through ALP activity, osteoblast-related gene expression, and mineralization, was stimulated by recombinant human FGF-2 (rhFGF-2) when applied during the proliferation phase, but not when applied during the differentiation stage alone. Our results suggest that FGF-2 indirectly promotes osteoblast differentiation as a downstream effect of stimulating cell proliferation. For the first time, we demonstrate that cyclic stretching induces MSCs to produce FGF-2, which in turn encourages cell proliferation through an autocrine/paracrine mechanism, consequently leading to osteoblast differentiation.
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Células Madre Mesenquimatosas , Osteogénesis , Humanos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Estrés Mecánico , Diferenciación Celular , Proliferación Celular , Osteoblastos/metabolismoRESUMEN
The underlying mechanisms of metastasis and recurrence of liver cancer remain largely unknown. Here, we found that Brother of the Regulator of Imprinted Sites (BORIS) variant SF2(C2/A4) was highly expressed in high metastatic potential hepatocellular carcinoma (HCC) cells and clinical tumor samples, related to the formation of satellite nodules. Its over expression promoted self-renewal, the expression of tumor stem cell markers, chemoresistance, wound healing rate, invasion and metastasis of HepG2 and Hep3B cells; reinforced epithelial-mesenchymal transition (EMT), decreased the expression of E-cadherin and increased N-cadherin and Vimentin. Subcellular localization experiment showed that BORIS SF2(C2/A4) was localized in nucleus and cytoplasm. Further double luciferase reporter gene experiment confirmed that it bound to TWIST1 gene promoter and significantly increased latter expression. BORIS SF2(C2/A4) knock down induced apoptosis of HCCLM3 and PLC/PRF/5 cells, and increased the protein content of cleaved caspase 3. Additionally, BORIS SF2(C2/A4) over expression increased the expression of fibroblast growth factor 2 (FGF2) in HepG2 and Hep3B cells. FGF2 expressed higher in HCC tumor tissues than in paired peri-tumor tissues, and its expression was positively correlated with BORIS SF2(C2/A4). Interestingly, high expression of FGF2 is also associated with the formation of satellite nodules. Moreover, using the medium from BORIS SF2(C2/A4) overexpressed cell lines to coculture hepatic stellate cell (HSCs) line LX-2, the latter could be activated and increased the expression of CD90 and PIGF, which is consistent with the effect of adding bFGF alone. These results indicate that BORIS SF2(C2/A4) plays a role in deterioration of liver cancer by regulating TWIST1 to induce EMT, and by FGF2 to activate HSCs.
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Carcinoma Hepatocelular , Proteínas de Unión al ADN , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Neoplasias Hepáticas/patología , Factor de Crecimiento Placentario/metabolismo , Proteínas de Unión al ADN/genéticaRESUMEN
Fibroblast growth factor 2 (FGF2) plays an important role in tumor angiogenesis. Humanized disulfide-stable double-chain antibody against fibroblast growth factor-2 (anti-FGF2 ds-Diabody) is a small molecule antibody with good tissue permeability and low immunogenicity, which has potential in tumor-targeted therapy. This study intended to investigate the effect of anti-FGF2 ds-Diabody on the migration and expression of programmed death-ligand1 (PD-L1) in hepatocellular carcinoma (HCC) cells. The anti-FGF2 ds-Diabody was expressed under methanol induction and purified with Ni2+ -affinity chromatography. Anti-FGF2 ds-Diabody significantly inhibited cell viability and proliferation in SK-Hep1 and HepG2 cells as confirmed by CCK-8 assays and colony formation assays. Western blot assays indicated that the proliferation of SK-Hep1 and HepG2 cells was inhibited by anti-FGF2 ds-Diabody through inhibiting the phosphorylation activation of AKT and MAPK. The results of transwell and western blot assays showed that the migration and invasion of SK-Hep1 and HepG2 cells were suppressed by anti-FGF2 ds-Diabody by affecting the epithelial-mesenchymal transition (EMT) process. Meanwhile, anti-FGF2 ds-Diabody inhibited the expression of PD-L1, and STAT3 participated in this process. Analysis of RT-PCR and Western blot suggested that fibroblast growth factor receptor 4 inhibitor 1 (FGFR4-IN-1) suppressed the expression of PD-L1, while STAT3 overexpression reversed this inhibitory effect. In addition, overexpression of STAT3 promoted migration and invasion and restored the suppressive effect of anti-FGF2 ds-Diabody on EMT. In conclusion, anti-FGF2 ds-Diabody could inhibit the expression of PD-L1 and EMT of hepatoma cells through FGF2/FGFR4/STAT3 axis. These results suggested that anti-FGF2 ds-Diabody has potential clinical application in inhibiting metastasis and immune escape of hepatocellular carcinoma.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Disulfuros/química , Transición Epitelial-Mesenquimal , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: Sepsis is defined as a life-threatening organ dysfunction caused by dysregulation of the host response to infection. There is still a lack of specific treatment for sepsis. Here, we report that Fibroblast growth factor-2 (FGF2) can reduce the mortality of sepsis by ameliorating the coagulation abnormalities. METHODS: FGF2 was intraperitoneally injected into septic mice induced by lipopolysaccharide (LPS) and then assessed for coagulation response, organ damage and survival. RAW264.7 cells with or without FGF2 pretreating were exposed to LPS, and then changes in coagulation related factors expression and signaling were tested. RESULTS: The findings showed that intraperitoneal injection of FGF2 inhibited coagulation activity, reduced lung and liver damage, and increased survival in septic mice. In RAW264.7 cells, LPS upregulated the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); however, pretreatment with FGF2 prevented this upregulation, while FGF2 knockdown exacerbated TF upregulation. Moreover, FGF2 suppressing the AKT/mTOR/S6K1 signaling pathway in septic mice and RAW264.7 cells stimulated by LPS. CONCLUSIONS: This study revealed a therapeutic role of FGF2 in ameliorating the coagulation abnormalities during sepsis.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos , Sepsis , Ratones , Animales , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Lipopolisacáridos/farmacología , Transducción de Señal , Coagulación SanguíneaRESUMEN
Osteoarthritis (OA) is a chronic debilitating degenerative disorder leading to structural, and functional anomaly of the joint. The present study tests the hypothesis that overexpression of the basic fibroblast growth factor (FGF-2) via direct rAAV-mediated gene transfer suppresses monosodium iodoacetate (MIA)-induced knee OA in rats relative to control (reporter rAAV-lacZ vector) gene transfer by intra-articular injection. Rats were treated with 20 µl rAAV-hFGF-2 on weekly basis; on days 7, 14, and 21 after single intra-articular injection of MIA (3 mg/50 µl saline). FGF-2 reduced knee joint swelling and improved motor performance and muscle coordination as evidenced by increased distance travelled, mean speed, rearing frequency in open field test (OFT) as well as fall-off latency in rotarod test together with reduced immobility time in OFT. Moreover, FGF-2 attenuated MIA-related radiological and histological alterations. Indeed, FGF-2 decreased knee joint inflammatory biomarker as demonstrated by reduced mRNA expression of toll like receptor (TLR)-4 and its downstream mediators such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß) and high motility group box (HMGB) 1. In parallel, FGF-2 attenuated knee joint degradation biomarkers as reflected by the downregulation of ADAMTS-5 mRNA expression and matrix metalloproteinase 13 (MMP-13) content together with the up-regulation of tissue inhibitor of metalloproteinase (TIMP)-1 mRNA expression. These findings suggest a potential therapeutic role for FGF-2 against MIA-induced knee OA in rats via inhibition of TLR4 signaling and activating TIMP-1, resulting in down-regulation of ADAMTS-5 and MMP-13.
Asunto(s)
Cartílago Articular , Osteoartritis , Animales , Ratas , Cartílago Articular/metabolismo , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/efectos adversos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Inyecciones Intraarticulares , Ácido Yodoacético , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Osteoartritis/patología , ARN Mensajero/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/uso terapéutico , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Humanos , Proteínas Recombinantes/farmacologíaRESUMEN
BACKGROUND: Apoptosis or programmed cell death plays a vital role in maintaining homeostasis and, therefore, is a tightly regulated process. Deregulation of apoptosis signalling can favour carcinogenesis. Apoptosis inhibitor 5 (Api5), an inhibitor of apoptosis, is upregulated in cancers. Interestingly, Api5 is shown to regulate both apoptosis and cell proliferation. To address the precise functional significance of Api5 in carcinogenesis here we investigate the role of Api5 in breast carcinogenesis. METHODS: Initially, we carried out in silico analyses using TCGA and GENT2 datasets to understand expression pattern of API5 in breast cancer patients followed by investigating the protein expression in Indian breast cancer patient samples. To investigate the functional importance of Api5 in breast carcinogenesis, we utilised MCF10A 3D breast acinar cultures and spheroid cultures of malignant breast cells with altered Api5 expression. Various phenotypic and molecular changes induced by altered Api5 expression were studied using these 3D culture models. Furthermore, in vivo tumorigenicity studies were used to confirm the importance of Api5 in breast carcinogenesis. RESULTS: In-silico analysis revealed elevated levels of Api5 transcript in breast cancer patients which correlated with poor prognosis. Overexpression of Api5 in non-tumorigenic breast acinar cultures resulted in increased proliferation and cells exhibited a partial EMT-like phenotype with higher migratory potential and disruption in cell polarity. Furthermore, during acini development, the influence of Api5 is mediated via the combined action of FGF2 activated PDK1-Akt/cMYC signalling and Ras-ERK pathways. Conversely, Api5 knock-down downregulated FGF2 signalling leading to reduced proliferation and diminished in vivo tumorigenic potential of the breast cancer cells. CONCLUSION: Taken together, our study identifies Api5 as a central player involved in regulating multiple events during breast carcinogenesis including proliferation, and apoptosis through deregulation of FGF2 signalling pathway.