RESUMEN
Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms.
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Citoesqueleto de Actina/metabolismo , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Células Madre Mesenquimatosas , Animales , Células Cultivadas , Femenino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Estudios ProspectivosRESUMEN
BACKGROUND: During last years, DNA vaccine immunogenicity has been optimized by the employment of co-stimulatory molecules and molecular adjuvants. It has been reported that plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the Anthrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates. Markedly, we faced with upsetting findings regarding the safety of pATRex as adjuvant since the aggregosome formation prompted to osteopenia in mice. OBJECTIVE: The present study provides additional evidences about the proteinaceous adjuvants action within bone marrow and questioned regarding the self-aggregation protein adjuvants immunotoxicity on marrow niches. METHODS & RESULTS: Using histological, biochemical and proteomic assays we shed light on pATRex effects within bone marrow niche and specifically we evidenced an aplastic-like bone marrow with disrupted cytokine/chemokine production. CONCLUSION: The above findings provide compelling support to the thesis that adjuvants based on plasmids encoding protein aggregation domains disrupt the physiological features of the bone marrow elements.
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Adyuvantes Inmunológicos/genética , Células de la Médula Ósea/metabolismo , Homeostasis/genética , Plásmidos/genética , Agregación Patológica de Proteínas/genética , Adyuvantes Inmunológicos/metabolismo , Animales , Sitios de Unión/genética , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , ADN/genética , ADN/inmunología , ADN/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Plásmidos/inmunología , Proteómica/métodosRESUMEN
IFN-γ is a pleotropic cytokine produced in the bone microenvironment. Although IFN-γ is known to play a critical role on bone remodeling, its function is not fully elucidated. Consistently, outcomes on the effects of IFN-γ recombinant protein on bone loss are contradictory among reports. In our work we explored, for the first time, the role of IFN-γ encoding plasmid (pIFN-γ) in a mouse model of osteopenia induced by ovariectomy and in the sham-operated counterpart to estimate its effects in skeletal homeostasis. Ovariectomy produced a dramatic decrease of bone mineral density (BMD). pINF-γ injected mice showed a pathologic bone and bone marrow phenotype; the disrupted cortical and trabecular bone microarchitecture was accompanied by an increased release of pro-inflammatory cytokine by bone marrow cells. Moreover, mesenchymal stem cells' (MSCs) commitment to osteoblast was found impaired, as evidenced by the decline of osterix-positive (Osx+) cells within the mid-diaphyseal area of femurs. For instance, a reduction and redistribution of CXCL12 cells have been found, in accordance with bone marrow morphological alterations. As similar effects were observed both in sham-operated and in ovariectomized mice, our studies proved that an increased IFN-γ synthesis in bone marrow might be sufficient to induce inflammatory and catabolic responses even in the absence of pathologic predisposing substrates. In addition, the obtained data might raise questions about pIFN-γ's safety when it is used as vaccine adjuvant.
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Densidad Ósea/efectos de los fármacos , Enfermedades Óseas Metabólicas/patología , Médula Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Interferón gamma/administración & dosificación , Animales , Enfermedades Óseas Metabólicas/metabolismo , Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células Cultivadas , Femenino , Ratones , OvariectomíaRESUMEN
The present study reports on the biocompatibility in vivo after intramuscular and subcutaneous administration in Balb/c mice of vinyl sulphone bearing p(HPMAm-lac1-2)-PEG-p(HPMAm-lac1-2)/thiolated hyaluronic acid hydrogels, designed as novel injectable biomaterials for potential application in the fields of tissue engineering and regenerative medicine. Ultrasonography, used as a method to study hydrogel gelation and residence time in vivo, showed that, upon injection, the biomaterial efficiently formed a hydrogel by simultaneous thermal gelation and Michael Addition cross-linking forming a viscoelastic spherical depot at the injection site. The residence time in vivo (20 days) was found to be shorter than that observed in vitro (32 days), indicating that the injected hydrogel was resorbed not only by chemical hydrolysis but also by cellular metabolism and/or enzymatic activity. Systemic biocompatibility was tested by analysing routine haematological parameters at different time-points (7, 14 and 21 days after administration) and histology of the main organs, including the haematopoietic system. No statistically significant difference between parameters of the saline-treated group and those of the hydrogel-treated group was found. Importantly, a time-dependent decrease of important pro-inflammatory cytokines (TREM1 (Triggering Receptor Expressed on Myeloid cells-1), tumour necrosis factor-α and interleukin-1ß) in cultured bone marrow cells extracted from hydrogel treated mice was observed, possibly correlated to the anti-inflammatory effect of hyaluronic acid released in time as hydrogel degraded. Copyright © 2016 John Wiley & Sons, Ltd.
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Citocinas/metabolismo , Ácido Hialurónico , Hidrogeles , Ensayo de Materiales , Polietilenglicoles , Animales , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Ratones , Ratones Endogámicos BALB C , Polietilenglicoles/química , Polietilenglicoles/farmacologíaRESUMEN
BACKGROUND: Plasmids coding protein aggregation polypeptides from different sources have been proposed as genetic adjuvants for DNA vaccines. We reported that a plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the Anthrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates and subsequent cell death. OBJECTIVE: In the present study we addressed the question of whether there is any substantial immunotoxicity associated with the use of self-aggregating proteins as genetic adjuvants. METHODS & RESULTS: Here we report, by mean of histology, X-ray and molecular examinations of bone specimens, the unexpected finding that intramuscular injection of pATRex in mice triggers, per se, severe bone loss (osteoporosis) independently from the sex and genotype of the treated animals. CONCLUSION: Even though the study suggests that proteinaceous "sticky " adjuvants are unlikely to find their way into practical vaccination, the information gained is of value as ATRex injections could provide an additional, simplified, mouse model of osteoporosis. Moreover, our results provide experimental support to the hypothesis that proteotoxic aggregates chronically activate the innate immune system in amyloid and aggregosome associated disorders.
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Enfermedades Óseas/terapia , ADN/genética , Plásmidos/genética , Animales , Enfermedades Óseas/genética , Modelos Animales de Enfermedad , Femenino , Fémur/diagnóstico por imagen , Fémur/patología , Inflamación/genética , Inflamación/terapia , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
We recently reported that a DNA plasmid coding p62-SQSTM1 acts as an effective anti tumor vaccine against both transplantable mouse tumors and canine spontaneous mammary neoplasms. Here we report the unexpected finding that intramuscular delivery of p62 DNA exerts a powerful anti-osteoporotic activity in a mouse model of inflammatory bone loss (i.e, ovariectomy) by combining bone-sparing and osteo-synthetic effects. Notably, the suppression of osteoporosis by p62DNA was associated with a sharp down-regulation of master inflammatory cytokines, and up-regulation of endogenous p62 protein by bone-marrow stromal cells. The present data provide a solid rational to apply p62 DNA vaccine as a safe, new therapeutic for treatment of inflammatory related bone loss diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Antiinflamatorios/farmacología , ADN/farmacología , Plásmidos/farmacología , Vacunas de ADN/farmacología , Animales , Antiinflamatorios/química , Clonación Molecular , ADN/genética , Femenino , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Distribución Aleatoria , Proteína Sequestosoma-1 , Vacunas de ADN/genéticaRESUMEN
The anatomical and functional dimensions of bone marrow topography have been at the forefront of modern bone and immunological research for many years and remain a source of complexity and perplexity due to the multitude of microhabitats within this microenvironment. In fact, research has uncovered fascinating functional aspects of bone marrow residents, and the bone marrow niche has been identified as the foremost reservoir of a variety of cells including hematopoietic, skeletal and endothelial stem/progenitor cells. The physical interactions of the marrow residents, combined with the release of cytokines and growth factors, organize well-defined operative compartments, which preserve bone and immune cell homeostasis. In a simplistic view, both the hematopoietic and bone marrow stromal (mesenchymal) stem/progenitor cell populations dwell at the interface between the endosteum and the bone marrow area (endosteal niche) and in the perivascular space (vascular niche). Indeed, the tantalizing hypothesis of bone marrow regulatory dependency on these niches is supported by current research insofar as the increase in the number of osteoblasts results in a concomitant increase in the hematopoietic population, indicating that the osteoblasts and the endosteal niche are key components of HSC maintenance. On the other hand, impaired function of the vascular niche compromises the endosteal niche's ability to support hematopoiesis. These fascinating discoveries indicate that there are strong ties between bone marrow inhabitants within the confines of the bone marrow itself. When these ties fail, niche-niche communication suffers and results in reduced bone formation, enfeebled hematopoiesis and unrestrained HSC migration through blood circulation. This study focused on the extraordinary homeostatic equilibrium and function of both bone and immune cells within the spatially defined microenvironment of bone marrow. But how important is the anatomically outlined scenery in which the bone marrow entity supports and hosts the hematopoietic elements?
Asunto(s)
Médula Ósea/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Remodelación Ósea , Homeostasis , Hormonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
Bone marrow mesenchymal stem cells (MSCs) have attracted considerable interest due to their ability to differentiate and contribute to the regeneration of mesenchymal tissues. The present study illustrates that the proper immobilization of heparin (Hep) and hyaluronic acid (HA) into a polypyrrole (PPy) matrix by electropolymerization results in an optimal interface for MSC differentiation towards osteoblast lineage. The obtained thin films showed good thermal stability, hydrophilicity and slow controlled polysaccharide release. The in vitro tests showed the main role of the interface chemical composition. Indeed, PPyHep and PPyHA thin films were able to induce osteogenic differentiation as determined by levels of specific early osteogenic markers (Runx2 and osterix) even in the absence of differentiating medium. Increased levels of ALP and Alizarin red staining, both indicating mineralization processes, confirmed the presence of mature osteoblasts.
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Huesos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Polímeros/química , Polisacáridos/farmacología , Pirroles/química , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Huesos/citología , Técnica del Anticuerpo Fluorescente , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos ICR , Polisacáridos/químicaRESUMEN
The microenvironment of bone marrow, an extraordinarily heterogeneous and dynamic system, is populated by bone and immune cells, and its functional dimension has been at the forefront of recent studies in the field of osteoimmunology. The interaction of both marrow niches supports self-renewal, differentiation, and homing of the hematopoietic stem cells and provides the essential regulatory molecules for osteoblast and osteoclast homeostasis. Impaired signaling within the niches results in a pathological tableau and enhances disease, including osteoporosis and arthritis, or the rejection of hematopoietic stem cell transplants. Discovering the anabolic players that control these mechanisms has become warranted. In this review, we focus on parathyroid hormone (PTH) and prostaglandins (PGs), potent molecular mediators, both of which carry out a multitude of functions, particularly in bone lining cells and T cells. These two regulators proved to be promising therapeutic agents when strictly clinical protocols on dose treatments were applied.
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Médula Ósea/inmunología , Huesos/inmunología , Sistema Inmunológico/fisiología , Hormona Paratiroidea/fisiología , Prostaglandinas/fisiología , Animales , Humanos , Hormona Paratiroidea/efectos adversos , Prostaglandinas/efectos adversos , Nicho de Células Madre/inmunología , Nicho de Células Madre/fisiología , Linfocitos T/inmunologíaRESUMEN
We previously generated separate lines of transgenic mice that specifically overexpress either the Fibroblast growth factor (FGF)-2 low-molecular-mass isoform (Tg(LMW)) or the high-mass isoforms (Tg(HMW)) in the osteoblast lineage. Vector/control (Tg(Vector)) mice were also made. Here we report the use of isolated calvarial osteoblasts (COBs) from those mice to investigate whether the FGF-2 protein isoforms differentially modulate bone formation in vitro. Our hypothesis states that FGF-2 isoforms specifically modulate bone morphogenetic protein 2 (BMP-2) function and subsequently bone differentiation genes and their related signaling pathways. We found a significant increase in alkaline phosphatase-positive colonies in Tg(LMW) COBs compared with Tg(Vector) controls. BMP-2 treatment significantly increased mineralized colonies in Tg(Vector) and Tg(LMW) COBs. BMP-2 caused a further significant increase in mineralized colonies in Tg(LMW) COBs compared with Tg(Vector) COBs but did not increase alkaline phosphatase-positive colonies in Tg(HMW) COBs. Time-course studies showed that BMP-2 caused a sustained increase in phosphorylated mothers against decapentaplegic-1/5/8 (Smad/1/5/8), runt-related transcription factor-2 (Runx-2), and osterix protein in Tg(LMW) COBs. BMP-2 caused a sustained increase in phospho-p38 MAPK in Tg(Vector) but only a transient increase in Tg(LMW) and Tg(HMW) COBs. BMP-2 caused a transient increase in phospho-p44/42 MAPK in Tg(Vector) COBs and no increase in Tg(LMW) COBs, but a sustained increase was found in Tg(HMW) COBs. Basal expression of FGF receptor 1 protein was significantly increased in Tg(LMW) COBs relative to Tg(Vector) COBs, and although BMP-2 caused a transient increase in FGF receptor 1 expression in Tg(Vector) COBs and Tg(HMW) COBs, there was no further increase Tg(LMW) COBs. Interestingly, although basal expression of FGF receptor 2 was similar in COBs from all genotypes, BMP-2 treatment caused a sustained increase in Tg(LMW) COBs but decreased FGF receptor 2 in Tg(Vector) COBs and Tg(HMW) COBs.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Cráneo/citología , Proteínas Smad/metabolismo , Factor de Transcripción Sp7 , Factores de Transcripción/metabolismoRESUMEN
Bone morphogenetic protein 2 (BMP-2) is one of the most potent regulators of osteoblast differentiation and bone formation. R-Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx-2 proteins have been shown to interact through their C-terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx-2 dependent, and Runx-2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP-2 up-regulated FGF-2 which is important for the maximal responses of BMP-2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP-2 induced nuclear accumulation and co-localization of Runx-2 and phospho-Smads1/5/8, while Runx/Smads nuclear accumulation and co-localization was reduced in Fgf2-/- osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx-2 in Fgf2-/- osteoblasts reduces R-Smads sub-nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Fluorescencia , Ratones , Osteoblastos/enzimología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cráneo/citología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Prostaglandin F2α (PGF2α) plays multiple roles on bone metabolism by regulating a wide range of signaling pathways. PGF2α, via activation of PKC, stimulates Na-dependent inorganic phosphate (Pi) transport system in osteoblasts; up-regulates interleukin (IL)-6 synthesis; increases vascular endothelial growth factor (VEGF). In addition, PGF2α acts as a strong mitogenic and survival agent on osteoblasts, and these effects are, at least in part, mediated by the binding of fibroblast growth factor-2 (FGF-2) to the specific receptor FGFR1. The understanding of PGF2α intracellular network, albeit complex to clarify, provides molecular bases useful to identify the players of osteoblast proliferation, apoptosis, and the associated angiogenic processes. Indeed, the molecular mechanism that underline PGF2α-regulated bone metabolism may be a promising platform for the development of novel targeted therapies in the treatment of bone disorders and disease.
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Remodelación Ósea/fisiología , Huesos/metabolismo , Dinoprost/metabolismo , Animales , Calcificación Fisiológica/fisiología , Dinoprost/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Homeostasis , Humanos , Interleucina-6/biosíntesis , Osteoblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Bone microenvironment is a complex dynamic equilibrium between osteoclasts and osteoblasts and is modulated by a wide variety of hormones and osteocyte mediators secreted in response to physiological and pathological conditions. The rate of remodeling involves tight coupling and regulation of both cells population and is regulated by a wide variety of hormones and mediators such as parathyroid hormone, prostaglandins, thyroid hormone, sex steroids, etc. It is also well documented that bone formation is easily influenced by the exposure of osteoblasts and osteoclasts to chemical compounds. Currently, humans and wildlife animals are exposed to various environmental xenoestrogens typically at low doses. These compounds, known as endocrine disruptor chemicals (EDCs), can alter the systemic hormonal regulation of the bone remodeling process and the skeletal formation. This review highlights the effects of the EDCs on mammalian bone turnover and development providing a macro and molecular view of their action.
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Remodelación Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Contaminantes Ambientales/toxicidad , Animales , Animales Salvajes , Remodelación Ósea/fisiología , Huesos/metabolismo , Microambiente Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ecosistema , Femenino , Humanos , Masculino , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , RatasRESUMEN
The present research examines the effects of 4-nonylphenol (4-NP) on mouse primary calvarial osteoblasts (COBs). Incubation of the cells with 4-NP at 10(-5)M and 10(-6)M striking decreased osteoblasts viability and phosphatidylserine (PS) exposure, measured by Annexin V, was greatly enhanced. In addition, an up-regulation of Bax/Bcl2 ratio with a drop in ΔΨm and an increase of cleaved caspase 9 and 3 was found, suggesting that the alkylphenol induced osteoblast death via the mitochondrial-dependent apoptotic pathway. Interestingly, treatment with 4-NP was also able to increase cleaved caspase 8 in parallel with the truncated active Bid (t-Bid) suggesting that 4-NP-mediated apoptosis depends on cross talk between the extrinsic and intrinsic pathways. It is of relevance, that the apoptotic effects of 4-NP overcame 17-ß-Estradiol (17-ß E(2)) induced-survival on osteoblasts. Also, the alkylphenol interfered with 17-ß E(2) regulated estrogen receptors expression.
Asunto(s)
Apoptosis/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Osteoblastos/efectos de los fármacos , Fenoles/toxicidad , Receptores de Estradiol/efectos de los fármacos , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/administración & dosificación , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Osteoblastos/metabolismo , Fenoles/administración & dosificación , Fosfatidilserinas/metabolismo , Receptores de Estradiol/metabolismo , Cráneo/citología , Cráneo/efectos de los fármacosRESUMEN
This study was performed to investigate the effects of the essential oil obtained from fruits of Ferulago campestris (FC) on primary calvarial mouse osteoblasts (COBs). The composition of the oil was dominated by monoterpene hydrocarbons (78.8-80.3%), with myrcene (33.4-39.7%), α-pinene (22.7-23.0%), and γ-terpinene (8.1-10.9%) as the major components. Owing to their lipophilic properties, these compounds easily cross cell membranes and affect bone cell function by stimulating or inhibiting specific molecular pathways. We demonstrated, for the first time, that FC oil increased osteoblast proliferation by MAP kinase activation; in addition, oils enhanced the protein kinase AKT, which is known to be critical for control of cell survival, also in presence of the MEK-1 inhibitor PD98059, and this effect was accompanied with a down-regulation of pro-apototic molecules such as Bax and caspases. Interestingly, FC oil significantly increased Runx2 (Runx2/Pebp2αA/AML3) and phospho-Smad1/5/8 protein level, the master regulators of osteoblast differentiation, and their nuclear localization. PD98059 pre-treatment further improved Runx2/phospho-Smads up-regulation. Thus, FC oils influence osteoblast metabolism probably using alternative signaling pathways depending also on the maturation stage of the cells. Taken together our data delineate a positive function of FC oil on osteoblast metabolism, suggesting its possible use as a dietetic integrator in the prevention or in the therapy of pathologies due to impaired bone remodeling.
Asunto(s)
Apiaceae/química , Aceites Volátiles/farmacología , Osteoblastos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromatografía de Gases , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Activación Enzimática , Cromatografía de Gases y Espectrometría de Masas , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína Smad5/metabolismoRESUMEN
Prostaglandin F2alpha (PGF2alpha) regulates fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor (FGFR) expression in osteoblasts. Here, the role of FGF-2 in PGF2alpha-induced proliferation and the signaling pathway involved, were determined in calvarial osteoblasts (COBs) from Fgf2+/+ and Fgf2-/- mice. The involvement of the exported FGF-2 isoform, was determined using the FGF-2 neutralizing antibody to alter its binding to FGFR1. PGF2alpha increased activity of Ras, and MAP-kinase cascade as well as Bcl-2 and c-Myc levels in Fgf2+/+ but not in Fgf2-/- COBs. Moreover, in Fgf2+/+ COBs, PGF2alpha-enhanced nuclear accumulation and co-localization of Bcl-2/c-Myc. Although up-regulation of multiple proliferative and survival signals were induced by PGF2alpha in Fgf2+/+ COBs, phospho-p53 was unmodified while p53 was increased. Increased phospho-p53 was, instead, found in Fgf2-/- COBs without up-regulation of oncogenic proteins. The lack of p53 activation in wild type osteoblasts could be due in part to the overexpression of MDM2 caused by PGF2alpha via FGF-2. PGF2alpha, also, increased cyclins D and E in Fgf2+/+ COBs and induced an expansion of Fgf2+/+ osteoblasts in G(2)/M phase. These data clearly show that PGF2alpha induces proliferation via endogenous FGF-2 and the exported isoform mediates PGF2alpha effects by acting in autocrine manner. Furthermore, silencing of FGFR1 in Fgf2+/+ COBs blocked PGF2alpha induced increase of phospho-MDM2 and cyclins.
Asunto(s)
Dinoprost/farmacología , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Proteínas de Ciclo Celular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoblastos/ultraestructura , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Cráneo/citología , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas ras/metabolismoRESUMEN
Parathyroid hormone (PTH) increases fibroblast growth factor receptor-1 (FGFR1) and fibroblast growth factor-2 (FGF-2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2-/- mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2-/- osteoblasts. PTH increased Runx-2 protein expression in Fgf2+/+ but not Fgf2-/- osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx-2 only in Fgf2+/+ osteoblasts. PTH and FGF-2 regulate Runx-2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho-CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2-/- osteoblasts. Silencing of FGF-2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx-2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1-cdk4/6 protein in Fgf2+/+ but not Fgf2-/- osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2-/- osteoblasts. PTH increased Bcl-2/Bax protein ratio in Fgf2+/+ but not Fgf2-/- osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2-/- osteoblasts. These data suggest that endogenous FGF-2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2-/- mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF-2 expression.
Asunto(s)
Anabolizantes/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Hormona Paratiroidea/farmacología , Anabolizantes/metabolismo , Animales , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Ratones , Ratones Noqueados , Osteoblastos/citología , Hormona Paratiroidea/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The present investigation extends our previous studies on PGF2alpha-mediated signalling in osteoblast metabolism. In particular, the role of PGF2alpha as modulator of heparan sulphate proteoglycans (HSPGs), fibroblast growth factor 2 (FGF-2) and fibroblast growth factor receptors (FGFRs) was evaluated. We hereby reported the novel observation that PGF2alpha was able to promote the formation of HSPGs/FGF-2/FGFRs complexes. Moreover, our data suggested that PGF2alpha could induce new synthesis of heparan sulphate (HS) chains on osteoblasts by a mechanism involving a modulation of MAPK signalling and that HS is required for the regulation of FGF-2 induced by PGF2alpha. Indeed, a proteolytic cleavage of HSPGs with heparinase III (Hep III) prior to PGF2alpha administration down-regulated the basal expression of phospho-p44/42, likely inhibiting FGFRs tyrosine kinase activity. Interestingly, MAPK signalling influenced syntheses and subcellular localization of FGF-2, its specific receptor and HS. In addition, the proteolytic cleavage by Hep III and the MAPK kinase inhibition by PD-98059 also revealed that PGF2alpha induced cell proliferation is dependent on HSPGs and FGF-2 specific receptor, respectively. Of further relevance of this study, we demonstrated, by using a specific siRNA for FGFR1, that PGF2alpha modulates Runx2 expression by FGFR1 and HS.
Asunto(s)
Dinoprost/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Western Blotting , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Microscopía Confocal , Microscopía Inmunoelectrónica , Interferencia de ARN , Transducción de Señal/fisiologíaRESUMEN
In this study, we investigated the role of prostaglandin F2alpha (PGF2alpha) in mouse osteoblast survival and the function of fibroblast growth factor 2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) in this process. In particular, for the first time, we demonstrated that PGF2alpha increased osteoblast survival in a dose-dependent manner and we showed that the effect is correlated with an increase in Bcl-2/Bax ratio. Furthermore, we demonstrated that PGF2alpha caused a decrement of the active caspases 9 and 3. By blocking FGF-2 with the specific neutralizing antibody and by depletion of FGFR1 gene with a specific siRNA, we showed that FGFR1 and FGF-2 are critical for the increment of Bcl-2/Bax ratio and the decrement of the active caspases 9 and 3, induced by PGF2alpha. Moreover, transmission electron microscopy studies showed that PGF2alpha increased binding of FGF-2 and FGFR1 and co-localization of reactive sites at plasma membrane level. In conclusion, we report a novel mechanism in which PGF2alpha induces FGF-2 binding to its specific cell surface receptor 1 leading to a cascade pathway that culminates with increased mouse osteoblast survival.
Asunto(s)
Apoptosis , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dinoprost/farmacología , Regulación hacia Abajo , Genes bcl-2 , Masculino , Ratones , Ratones Endogámicos ICR , Interferencia de ARN , Cráneo/citología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
We previously reported that transient administration of phthalates induced actin cytoskeleton disruption in Py1a osteoblasts. However, the mechanism of this transient effect was not elucidated. In this study we provided evidence that the actin cytoskeletal re-established conditions are dependent on new actin expression and synthesis. To assess the role of phthalates in modulating the distribution of actin, confocal and electron microscopy studies were carried out. Results indicated a modification of actin distribution after phthalate administration. In addition, a relation with the nucleoskeletal component lamin A supports the hypothesis that phthalates may participate in regulatory cell processes involving actin in Py1a osteoblasts. The present study also supports the mitogenic effects of phthalates, which involve microfilament disruption, nuclear actin and lamin A. In particular, the increased levels of cyclin D3, which in mammalian cells plays a critical role in G1 to S transition and is a putative proto-oncogene in benzyl butyl phthalate treated cells, suggested a possible effect of the endocrine disruptor in cancer processes.