RESUMEN
BACKGROUND: Osteoclasts are major actors in the maintenance of bone homeostasis. The full functional maturation of osteoclasts from monocyte lineage cells is essential for the degradation of old/damaged bone matrix. Diuron is one of the most frequently encountered herbicides, particularly in water sources. However, despite a reported delayed ossification in vivo, its impact on bone cells remains largely unknown. OBJECTIVES: The objectives of this study were to first better characterize osteoclastogenesis by identifying genes that drive the differentiation of CD14+ monocyte progenitors into osteoclasts and to evaluate the toxicity of diuron on osteoblastic and osteoclastic differentiation in vitro. METHODS: We performed chromatin immunoprecipitation (ChIP) against H3K27ac followed by ChIP-sequencing (ChIP-Seq) and RNA-sequencing (RNA-Seq) at different stages of differentiation of CD14+ monocytes into active osteoclasts. Differentially activated super-enhancers and their potential target genes were identified. Then to evaluate the toxicity of diuron on osteoblasts and osteoclasts, we performed RNA-Seq and functional tests during in vitro osteoblastic and osteoclastic differentiation by exposing cells to different concentrations of diuron. RESULTS: The combinatorial study of the epigenetic and transcriptional remodeling taking place during differentiation has revealed a very dynamic epigenetic profile that supports the expression of genes vital for osteoclast differentiation and function. In total, we identified 122 genes induced by dynamic super-enhancers at late days. Our data suggest that high concentration of diuron (50µM) affects viability of mesenchymal stem cells (MSCs) in vitro associated with a decrease of bone mineralization. At a lower concentration (1µM), an inhibitory effect was observed in vitro on the number of osteoclasts derived from CD14+ monocytes without affecting cell viability. Among the diuron-affected genes, our analysis suggests a significant enrichment of genes targeted by pro-differentiation super-enhancers, with an odds ratio of 5.12 (ρ=2.59×10-5). DISCUSSION: Exposure to high concentrations of diuron decreased the viability of MSCs and could therefore affect osteoblastic differentiation and bone mineralization. This pesticide also disrupted osteoclasts maturation by impairing the expression of cell-identity determining genes. Indeed, at sublethal concentrations, differences in the expression of these key genes were mild during the course of in vitro osteoclast differentiation. Taken together our results suggest that high exposure levels of diuron could have an effect on bone homeostasis. https://doi.org/10.1289/EHP11690.
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Herbicidas , Osteogénesis , Humanos , Diurona , Secuencias Reguladoras de Ácidos Nucleicos , Diferenciación CelularRESUMEN
In bone regeneration induced by the combination of mesenchymal stromal cells (MSCs) and calcium-phosphate (CaP) materials, osteoclasts emerge as a pivotal cell linking inflammation and bone formation. Favorable outcomes are observed despite short-term engraftments of implanted MSCs, highlighting their major paracrine function and the possible implication of cell death in modulating their secretions. In this work, we focused on the communication from MSCs towards osteoclasts-like cells in vitro. MSCs seeded on a CaP biomaterial or undergoing induced apoptosis produced a conditioned media favoring the development of osteoclasts from human CD14+ monocytes. On the contrary, MSCs' apoptotic secretion inhibited the development of inflammatory multinucleated giant cells formed after IL-4 stimulation. Components of MSCs' secretome before and after apoptotic stress were compared using mass spectrometry-based quantitative proteomics and a complementary immunoassay for major cytokines. CXCR-1 and CXCR-2 ligands, primarily IL-8/CXCL-8 but also the growth-regulated proteins CXCL-1, -2 or -3, were suggested as the major players of MSCs' pro-osteoclastic effect. These findings support the hypothesis that osteoclasts are key players in bone regeneration and suggest that apoptosis plays an important role in MSCs' effectiveness.
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Apoptosis , Células de la Médula Ósea/citología , Diferenciación Celular , Células Gigantes/patología , Células Madre Mesenquimatosas/citología , Osteoclastos/citología , Osteogénesis , Células de la Médula Ósea/fisiología , Proliferación Celular , Citocinas , Células Gigantes/metabolismo , Humanos , Células Madre Mesenquimatosas/fisiología , Osteoclastos/fisiologíaRESUMEN
Osteosarcoma (OS) is the most common form of primary bone tumor affecting mainly children and young adults. Despite therapeutic progress, the 5-year survival rate is 70%, but it drops drastically to 30% for poor responders to therapies or for patients with metastases. Identifying new therapeutic targets is thus essential. Heat Shock Proteins (HSPs) are the main effectors of Heat Shock Response (HSR), the expression of which is induced by stressors. HSPs are a large family of proteins involved in the folding and maturation of other proteins in order to maintain proteostasis. HSP overexpression is observed in many cancers, including breast, prostate, colorectal, lung, and ovarian, as well as OS. In this article we reviewed the significant role played by HSPs in molecular mechanisms leading to OS development and progression. HSPs are directly involved in OS cell proliferation, apoptosis inhibition, migration, and drug resistance. We focused on HSP27, HSP60, HSP70 and HSP90 and summarized their potential clinical uses in OS as either biomarkers for diagnosis or therapeutic targets. Finally, based on different types of cancer, we consider the advantage of targeting heat shock factor 1 (HSF1), the major transcriptional regulator of HSPs in OS.
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Neoplasias Óseas/diagnóstico , Neoplasias Óseas/terapia , Chaperonas Moleculares/metabolismo , Osteosarcoma/diagnóstico , Osteosarcoma/terapia , Animales , Neoplasias Óseas/metabolismo , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Modelos Biológicos , Osteosarcoma/metabolismoRESUMEN
Osteosarcoma (OS) is the most common primary bone tumor, mainly occurring in children and adolescents. Current standard therapy includes tumor resection associated with multidrug chemotherapy. However, patient survival has not evolved for the past decades. Since the 1970s, the 5-year survival rate is around 75% for patients with localized OS but dramatically drops to 20% for bad responders to chemotherapy or patients with metastases. Resistance is one of the biological processes at the origin of therapeutic failure. Therefore, it is necessary to better understand and decipher molecular mechanisms of resistance to conventional chemotherapy in order to develop new strategies and to adapt treatments for patients, thus improving the survival rate. This review will describe most of the molecular mechanisms involved in OS chemoresistance, such as a decrease in intracellular accumulation of drugs, inactivation of drugs, improved DNA repair, modulations of signaling pathways, resistance linked to autophagy, disruption in genes expression linked to the cell cycle, or even implication of the micro-environment. We will also give an overview of potential therapeutic strategies to circumvent resistance development.
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Current therapeutic approaches to osteoporosis display some potential adverse effects and a limited efficacy on non-vertebral fracture reduction. Some sulfonylamidines targeting the scaffold proteins prohibitins-1 and 2 (PHB1/2) have been showed to inhibit the formation of osteoclasts in charge of bone resorption. Herein, we report the development of a second generation of anti-osteoclastic PHB ligands. The most potent compound, IN45, showed 88% inhibition at the low concentration of 5 µM, indicates that it might serve as a basis for the development of new antiosteoporotic drugs.
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Amidinas/química , Amidinas/farmacología , Osteogénesis/efectos de los fármacos , Proteínas Represoras/metabolismo , Células Cultivadas , Descubrimiento de Drogas , Humanos , Ligandos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Prohibitinas , Compuestos de Azufre/química , Compuestos de Azufre/farmacologíaRESUMEN
Ribosomopathies are a group of rare diseases in which genetic mutations cause defects in either ribosome biogenesis or function, given specific phenotypes. Ribosomal proteins, and multiple other factors that are necessary for ribosome biogenesis (rRNA processing, assembly of subunits, export to cytoplasm), can be affected in ribosomopathies. Despite the need for ribosomes in all cell types, these diseases result mainly in tissue-specific impairments. Depending on the type of ribosomopathy and its pathogenicity, there are many potential therapeutic targets. The present manuscript will review our knowledge of ribosomopathies, discuss current treatments, and introduce the new therapeutic perspectives based on recent research. Diamond-Blackfan anemia, currently treated with blood transfusion prior to steroids, could be managed with a range of new compounds, acting mainly on anemia, such as L-leucine. Treacher Collins syndrome could be managed by various treatments, but it has recently been shown that proteasomal inhibition by MG132 or Bortezomib may improve cranial skeleton malformations. Developmental defects resulting from ribosomopathies could be also treated pharmacologically after birth. It might thus be possible to treat certain ribosomopathies without using multiple treatments such as surgery and transplants. Ribosomopathies remain an open field in the search for new therapeutic approaches based on our recent understanding of the role of ribosomes and progress in gene therapy for curing genetic disorders.
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Anemia de Diamond-Blackfan/terapia , Proteínas Ribosómicas/genética , Ribosomas/genética , Humanos , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers. METHODS: The aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases. FINDINGS: MTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90â¯mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases. INTERPRETATION: All these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma. FUNDING: This study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé.
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Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Pirimidinas/uso terapéutico , 8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Neoplasias Óseas/patología , Línea Celular Tumoral , ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Humanos , Neoplasias Pulmonares/secundario , Ratones , Osteosarcoma/patología , Monoéster Fosfórico Hidrolasas/metabolismo , Pirimidinas/farmacología , Células Tumorales CultivadasRESUMEN
Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development.
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Enfermedades del Desarrollo Óseo/genética , Enanismo/genética , Mutación Missense/genética , Proteínas de Neoplasias/genética , Proteínas Ribosómicas/genética , Anemia de Diamond-Blackfan/genética , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Vascular calcifications are associated with a high cardiovascular morbi-mortality in the coronary territory. In parallel, femoral arteries are more calcified and develop osteoid metaplasia (OM). This study was conducted to assess the predictive value of OM and local inflammation on the occurrence of mid- and long-term adverse cardiovascular events. METHOD: Between 2008 and 2015, 86 atheromatous samples were harvested during femoral endarterectomy on 81 patients and processed for histomorphological analyses of calcifications and inflammation (monocytes and B cells). Histological findings were compared with the long-term follow-up of patients, including major adverse cardiac event (MACE), major adverse limb event (MALE), and mortality. Frequencies were presented as percentage, and continuous data, as mean and standard deviation. A P-value < 0.05 was considered statistically significant. RESULTS: Median follow-up was 42.4 months (26.9-58.8). Twenty-eight percent of patients underwent a MACE; a MALE occurred in 18 (21%) limbs. Survival rate was 87.2% at 36 months. OM was found in 41 samples (51%), without any significant impact on the occurrence of MACE, MALE, or mortality. Preoperative white blood cell formulae revealed a higher rate of neutrophils associated with MACE (P = 0.04) and MALE (P = 0.0008), correlated with higher B cells counts in plaque samples. CONCLUSIONS: OM is part of femoral calcifications in almost 50% of the cases but does not seem to be an independent predictive variable for MACE or MALE. However, a higher rate of B cell infiltration of the plaque and preoperative neutrophil blood count may be predictive of adverse events during follow-up.
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Arteria Femoral/patología , Osificación Heterotópica , Enfermedad Arterial Periférica/patología , Calcificación Vascular/patología , Anciano , Anciano de 80 o más Años , Amputación Quirúrgica , Linfocitos B/inmunología , Linfocitos B/patología , Progresión de la Enfermedad , Endarterectomía , Femenino , Arteria Femoral/inmunología , Arteria Femoral/cirugía , Francia/epidemiología , Humanos , Recuperación del Miembro , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Neutrófilos/patología , Enfermedad Arterial Periférica/inmunología , Enfermedad Arterial Periférica/mortalidad , Enfermedad Arterial Periférica/cirugía , Placa Aterosclerótica , Reoperación , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Calcificación Vascular/inmunología , Calcificación Vascular/mortalidad , Calcificación Vascular/cirugíaRESUMEN
The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE ) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes .
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Huesos/citología , Hibridación in Situ/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Línea Celular Tumoral , ADN/genética , ADN/aislamiento & purificación , Digoxigenina/química , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Técnicas de Preparación Histocitológica/instrumentación , Técnicas de Preparación Histocitológica/métodos , Humanos , Hibridación in Situ/instrumentación , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/aislamiento & purificación , Oligonucleótidos/química , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: Krenn synovitis Score has been developed by Krenn et al. in order to assess synovitis severity and is used in synovial research. Cell signature of synovial tissue can be studied using immunohistochemistry and is of interest as a biomarker for both prognosis and prediction of response to treatment. However, no synovitis score including immunohistochemistry exists yet. In order to answer this unmet need, we propose a new Immunologic Synovitis score (IMSYC) adding 5 components to the Krenn score: CD68, CD3, CD20, CD31 and Ki67 immunostaining. In this study, we aimed to validate this new IMSYC by studying its diagnostic performances in a well-defined collection of synovial samples. METHODS: Synovial samples from patients were obtained during surgical procedures. CD68, CD3, CD20, CD31 and KI67 immunohistochemistry were performed. RESULTS: In total, 77 patients were included. In total, 45 were females, mean age was 63.1 years. Forty had inflammatory arthritis, mainly rheumatoid arthritis (31/40). Non inflammatory arthritis group included 35 patients with mainly osteoarthritis. Mean Krenn score and IMSYC were significantly higher in the inflammatory group (P<0.001). ROC analysis of diagnostic performances determined the score of 13.5 out of 24 as the cut-off that gave the best ratio for discrimination between inflammatory and non-inflammatory arthritis with a sensitivity of 71.8% and specificity of 98%. CONCLUSION: We propose a new synovitis score including immunohistochemistry. This score has a better sensitivity and specificity than the Krenn score and represents a more functional synovitis evaluation. IMSYC could be further used in better categorizing synovial tissue phenotype and give a basis for tissue driven therapy.
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Artritis Reumatoide/inmunología , Índice de Severidad de la Enfermedad , Membrana Sinovial/inmunología , Sinovitis/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , SinovectomíaRESUMEN
Vascular calcification is a strong and independent predictive factor for cardiovascular complications and mortality. Our previous work identified important discrepancies in plaque composition and calcification types between carotid and femoral arteries. The objective of this study is to further characterize and understand the heterogeneity in vascular calcification among vascular beds, and to identify molecular mechanisms underlying this process. We established ECLAGEN biocollection that encompasses human atherosclerotic lesions and healthy arteries from different locations (abdominal, thoracic aorta, carotid, femoral, and infrapopliteal arteries) for histological, cell isolation, and transcriptomic analysis. Our results show that lesion composition differs between these locations. Femoral arteries are the most calcified arteries overall. They develop denser calcifications (sheet-like, nodule), and are highly susceptible to osteoid metaplasia. These discrepancies may derive from intrinsic differences between SMCs originating from these locations, as microarray analysis showed specific transcriptomic profiles between primary SMCs isolated from each arterial bed. These molecular differences translated into functional disparities. SMC from femoral arteries showed the highest propensity to mineralize due to an increase in basal TGFß signaling. Our results suggest that biological heterogeneity of resident vascular cells between arterial beds, reflected by our transcriptomic analysis, is critical in understanding plaque biology and calcification, and may have strong implications in vascular therapeutic approaches.
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Arterias/patología , Calcinosis/patología , Músculo Liso Vascular/patología , Diferenciación Celular , Células Cultivadas , Humanos , Placa Aterosclerótica/patología , TranscriptomaRESUMEN
Gap junctions are transmembrane structures that directly connect the cytoplasm of adjacent cells, making intercellular communications possible. It has been shown that the behaviour of several tumours - such as bone tumours - is related to gap junction intercellular communications (GJIC). Several methodologies are available for studying GJIC, based on measuring different parameters that are useful for multiple applications, such as the study of carcinogenesis for example. These methods nevertheless have several limitations. The present manuscript describes the setting up of a dielectrophoresis (DEP)-based lab-on-a-chip platform for the real-time study of Gap Junctional Intercellular Communication between osteosarcoma cells and the main cells accessible to their microenvironment. We conclude that using the DEParray technology for the GJIC assessment has several advantages comparing to current techniques. This methodology is less harmful for cells integrity; cells can be recovered after interaction to make further molecular analysis; it is possible to study GJIC in real time; we can promote cell interactions using up to five different populations. The setting up of this new methodology overcomes several difficulties to perform experiments for solving questions about GJIC process that we are not able to do with current technics.
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Comunicación Celular/fisiología , Electroforesis por Microchip/instrumentación , Electroforesis por Microchip/métodos , Uniones Comunicantes/fisiología , Neoplasias Óseas/patología , Diferenciación Celular/fisiología , Línea Celular Tumoral , Humanos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteosarcoma/patología , Imagen de Lapso de Tiempo/métodosRESUMEN
Histone modifications are important for maintaining the transcription program. BET proteins, an important class of "histone reading proteins", have recently been described as essential in bone biology. This study presents the therapeutic opportunity of BET protein inhibition in osteoporosis. We find that the pharmacological BET protein inhibitor JQ1 rescues pathologic bone loss in a post-ovariectomy osteoporosis model by increasing the trabecular bone volume and restoring mechanical properties. The BET protein inhibition suppresses osteoclast differentiation and activity as well as the osteoblastogenesis in vitro. Moreover, we show that treated non-resorbing osteoclasts could still activate osteoblast differentiation. In addition, specific inhibition of BRD4 using RNA interference inhibits osteoclast differentiation but strongly activates osteoblast mineralization activity. Mechanistically, JQ1 inhibits expression of the master osteoclast transcription factor NFATc1 and the transcription factor of osteoblast Runx2. These findings strongly support that targeting epigenetic chromatin regulators such as BET proteins may offer a promising alternative for the treatment of bone-related disorders such as osteoporosis.
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Epigénesis Genética , Proteínas Nucleares/antagonistas & inhibidores , Osteoporosis/tratamiento farmacológico , Osteoporosis/genética , Transducción de Señal , Animales , Azepinas/farmacología , Azepinas/uso terapéutico , Fenómenos Biomecánicos , Recuento de Células , Diferenciación Celular , Epigénesis Genética/efectos de los fármacos , Femenino , Humanos , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteoporosis/patología , Osteoporosis/fisiopatología , Ovariectomía , Transducción de Señal/efectos de los fármacos , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
IL-34 is a proinflammatory cytokine implicated in rheumatoid arthritis (RA). The current study aimed to assess the IL-34 expression in response to two members of the transforming growth factor (TGF)-ß family, TGF-ß1 and bone morphogenetic protein (BMP)-2, in synovial fibroblasts from RA patients. IL-34, TGF-ß1, and BMP-2 productions were measured in patient synovial fluids by enzyme-linked immunosorbent assay. IL-34 mRNA levels were quantified by real-time quantitative PCR in human synovial fibroblasts and murine mesenchymal stem cells. Pharmacologic inhibitions were used to determine the involvement of activin receptor-like kinase 1 (ALK1) and ALK5 downstream TGF-ß1 and BMP-2. IL-34, TGF-ß1, and BMP-2 were expressed in synovial fluids from RA patients. We found a significant correlation between IL-34 and TGF-ß1 expressions. Levels of both IL-34 and TGF-ß1 were thus correlated with the total leukocyte counts in the synovial fluids. TGF-ß1 and BMP-2 decreased IL-34 expression in the synovial fibroblasts or in murine mesenchymal stem cells in a dose- and time-dependent manner through ALK5 and ALK1 pathways, respectively. In addition, TGF-ß1 and BMP-2 antagonized tumor necrosis factor α-induced IL-34 gene expression. This work identifies TGF-ß1 and BMP-2 as potent inhibitors of IL-34 expression in RA synovial fibroblasts. These cytokines, as upstream inhibitors of IL-34, may thus contribute to antagonize inflammation and bone erosions in RA.
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Artritis Reumatoide/patología , Proteína Morfogenética Ósea 2/metabolismo , Fibroblastos/metabolismo , Inflamación/patología , Interleucinas/metabolismo , Membrana Sinovial/patología , Factor de Crecimiento Transformador beta1/metabolismo , Receptores de Activinas Tipo II/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Fibroblastos/patología , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Persona de Mediana Edad , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
OBJECTIVES: The effect of amino-bisphosphonates on osteoblastic lineage and its potential contribution to the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw (BONJ) remain controversial. We assessed the effects of zoledronic acid (ZOL) on bone and vascular cells of the alveolar socket using a mouse model of BONJ. MATERIAL AND METHODS: Thirty-two mice were treated twice a week with either 100 µg/kg of ZOL or saline for 12 weeks. The first left maxillary molar was extracted at the third week. Alveolar sockets were assessed at both 3 weeks (intermediate) and 9 weeks (long-term) after molar extraction by semi-quantitative histomorphometry for empty lacunae, preosteoblasts (Osterix), osteoclasts (TRAP), and pericyte-like cells (CD146). Also, the bone microarchitecture was assessed by micro-CT. RESULTS: Osteonecrotic-like lesions were observed in 21% of mice. Moreover, a decreased number of preosteoblasts contrasted with the increased number of osteoclasts at both time points. In addition, osteoclasts display multinucleation and detachment from the endosteal surface. Furthermore, the number of pericyte-like cells increased at the intermediate time point. The alveolar bone mass increased exclusively with long-term ZOL treatment. CONCLUSION: The severe imbalance between bone-forming cells and bone-resorbing cells shown in this study could contribute to the pathogenesis of BONJ.
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Osteonecrosis de los Maxilares Asociada a Difosfonatos/patología , Conservadores de la Densidad Ósea/toxicidad , Difosfonatos/toxicidad , Imidazoles/toxicidad , Osteoblastos/patología , Alveolo Dental/efectos de los fármacos , Animales , Conservadores de la Densidad Ósea/administración & dosificación , Diferenciación Celular , Difosfonatos/administración & dosificación , Modelos Animales de Enfermedad , Imidazoles/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Diente Molar/cirugía , Extracción Dental , Microtomografía por Rayos X , Ácido ZoledrónicoRESUMEN
Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an antimetastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFß-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFß effectors, SMAD4 and TßRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound-healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFß-dependent signaling node that switches off antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.
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Neoplasias Óseas/patología , Neoplasias Pulmonares/secundario , MicroARNs/antagonistas & inhibidores , Osteosarcoma/patología , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Cicatrización de Heridas , Animales , Apoptosis , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Movimiento Celular , Proliferación Celular , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , MicroARNs/genética , Osteosarcoma/genética , Osteosarcoma/metabolismo , Isoformas de Proteínas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Activación Transcripcional , Factor de Crecimiento Transformador beta/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin-34 (IL-34) is a newly-discovered homodimeric cytokine that regulates, like Macrophage Colony-Stimulating Factor (M-CSF), the differentiation of the myeloid lineage through M-CSF receptor (M-CSFR) signaling pathways. To date, both cytokines have been considered as competitive cytokines with regard to the M-CSFR. The aim of the present work was to study the functional relationships of these cytokines on cells expressing the M-CSFR. We demonstrate that simultaneous addition of M-CSF and IL-34 led to a specific activation pattern on the M-CSFR, with higher phosphorylation of the tyrosine residues at low concentrations. Similarly, both cytokines showed an additive effect on cellular proliferation or viability. In addition, BIAcore experiments demonstrated that M-CSF binds to IL-34, and molecular docking studies predicted the formation of a heteromeric M-CSF/IL-34 cytokine. A proximity ligation assay confirmed this interaction between the cytokines. Finally, co-expression of the M-CSFR and its ligands differentially regulated M-CSFR trafficking into the cell. This study establishes a new foundation for the understanding of the functional relationship between IL-34 and M-CSF, and gives a new vision for the development of therapeutic approaches targeting the IL-34/M-CSF/M-CSFR axis.
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Citocinas/fisiología , Interleucinas/química , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/química , Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Supervivencia Celular , Citocinas/química , Células HEK293 , Humanos , Interleucinas/farmacología , Simulación del Acoplamiento Molecular , Monocitos/fisiología , Fosforilación , Multimerización de Proteína , Transducción de SeñalRESUMEN
Receptor activating NF-κB ligand (RANKL) is a member of the TNF superfamily that plays a pivotal role in bone homeostasis as being the major osteoclastogenesis factor. RANKL also has pleiotropic effects in the immune system in which it is expressed by activated T and B cells and some innate lymphoid cells. RANKL-RANK interactions mediate lymph node organogenesis and immunoregulatory functions in autoimmune disease and carcinogenesis as well as cross talk between the immune system and bone. In this study, we show that basophils were the strongest RANKL mRNA-expressing cells amongst major leukocyte subsets in human blood. RANKL was preformed as an intracellular protein in resting basophils and was rapidly and strongly expressed on their surface upon stimulation with IL-3, but not other stimuli. This expression was stable for at least 6 days. Activated basophils could also release soluble RANKL in small quantities upon interaction with DCs or monocytes. In the blood, basophils were the sole cells to express membrane RANKL in response to IL-3. This study indicates that basophils should be considered as new players in the pleiotropic and complex RANKL-RANK interaction system and suggests a role for RANKL in the interaction between basophils and immune cells in inflammatory allergic tissues and secondary lymphoid organs.