RESUMEN
Zingerone, 4-(4-hydroxy-3-methoxyphenyl)-2-butanone (Zg), a phenolic compound isolated from ginger is reported to have anti-inflammatory and antidiabetic properties. However, its role in the promotion of osteogenesis is not known. In this study, we investigated the therapeutic effect of Zg on osteogenesis at the cellular and molecular levels. Zg treatment was nontoxic to mouse mesenchymal stem cells (mMSCs). At the cellular level, it enhanced osteoblast differentiation as evidenced by more calcium deposits. At the molecular level, Zg stimulated the expression of Runx2 (a bone transcription factor) and other marker genes of osteoblast differentiation in mMSCs. Recent studies indicated that microRNAs (miRNAs) regulate bone metabolism, and we identified that Zg treatment in mMSCs upregulated mir-590, a positive regulator of Runx2 by targeting Smad7, an antagonist of TGF-ß1 signaling. Thus, the osteogenic potential of Zg would be beneficial for treating bone and bone-related diseases.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Guayacol/análogos & derivados , MicroARNs/genética , Osteogénesis/efectos de los fármacos , Animales , Conservadores de la Densidad Ósea/aislamiento & purificación , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Zingiber officinale/química , Guayacol/aislamiento & purificación , Guayacol/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , MicroARNs/agonistas , MicroARNs/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/genética , Transducción de Señal , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Endocrine disorders have become more and more frequently diagnosed in humans and animals. In horses, equine metabolic syndrome (EMS) is characterized by insulin resistance, hyperleptinemia, hyperinsulinemia, inflammation and usually by pathological obesity. Due to an increased inflammatory response in the adipose tissue, cytophysiological properties of adipose derived stem cells (ASC) have been impaired, which strongly limits their therapeutic potential. Excessive accumulation of reactive oxygen species, mitochondria deterioration and accelerated ageing of those cells affect their multipotency and restrict the effectiveness of the differentiation process. In the present study, we have treated ASC isolated from EMS individuals with a combination of 5-azacytydine (AZA) and resveratrol (RES) in order to reverse their aged phenotype and enhance osteogenic differentiation. Using SEM and confocal microscope, cell morphology, matrix mineralization and mitochondrial dynamics were assessed. Furthermore, we investigated the expression of osteogenic-related genes with RT-PCR. We also investigated the role of autophagy during differentiation and silenced PARKIN expression with siRNA. Obtained results indicated that AZA/RES significantly enhanced early osteogenesis of ASC derived from EMS animals. Increased matrix mineralization, RUNX-2, collagen type I and osteopontin levels were noted. Furthermore, we proved that AZA/RES exerts its beneficial effects by modulating autophagy and mitochondrial dynamics through PARKIN and RUNX-2 activity.
Asunto(s)
Azacitidina/farmacología , Enfermedades de los Caballos/tratamiento farmacológico , Células Madre Mesenquimatosas/efectos de los fármacos , Síndrome Metabólico/veterinaria , Obesidad/veterinaria , Osteogénesis/efectos de los fármacos , Resveratrol/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Diferenciación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Combinación de Medicamentos , Femenino , Regulación de la Expresión Génica , Enfermedades de los Caballos/genética , Enfermedades de los Caballos/patología , Caballos , Resistencia a la Insulina , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/genética , Síndrome Metabólico/patología , Dinámicas Mitocondriales/efectos de los fármacos , Obesidad/tratamiento farmacológico , Obesidad/genética , Obesidad/patología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/genética , Osteopontina/agonistas , Osteopontina/genética , Osteopontina/metabolismo , Estrés Oxidativo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Bone homeostasis is maintained by balancing bone formation and bone resorption, but an imbalance between them is associated with various bone-related diseases such as osteoporosis and rheumatoid arthritis. We found that 5,6-dehydrokawain (DK) and dihydro-5,6-dehydrokawain (DDK), which were isolated as promising compounds from Alpinia zerumbet rhizomes, promote differentiation of osteoblastic MC3T3-E1 cells. DK and DDK increased the alkaline phosphatase activity and matrix mineralization of MC3T3-E1 cells. DK exerts larger effects than DDK. The gene expression of runt-related transcription factor 2 and osterix, which are essential transcription factors in the early period of osteoblast differentiation, was significantly increased by DK treatment. The mRNA level of distal-less homeobox 5 was also enhanced by DK treatment, and DK activated the p38 mitogen-activated protein kinase pathway. Therefore, DK may have clinical potential for preventing osteoporosis, and could be considered as a potential anabolic therapeutic agent.
Asunto(s)
Alpinia/química , Diferenciación Celular/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Pironas/farmacología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/agonistas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/genética , Extractos Vegetales/química , Pironas/aislamiento & purificación , ARN Mensajero/agonistas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rizoma/química , Factor de Transcripción Sp7 , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
miRNAs play a number of roles in bone, including mediating the pathological effects of inflammation. Here, we found that miR-33a-5p expression was significantly increased after TNF-α treatment during BMP-2-induced osteogenic differentiation of hBMSCs. Luciferase reporter assays and western blotting demonstrated that special AT-rich sequence-binding protein 2 (SATB2) is a target of miR-33a-5p. Moreover, we show that BMP-2 induces SATB2 expression by interacting with SATB2 directly via the BMP-2-RUNX2 pathway. However, TNF-α first decreases SATB2 expression by inhibiting miR-33a-5p degradation. We thus conclude that miR-33a-5p plays a central role in this complex regulatory network. These findings will help to understand the regulatory role of miR-33a-5p in the inflammatory process.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , MicroARNs/metabolismo , Osteogénesis , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteína Morfogenética Ósea 2/genética , Proliferación Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Humanos , Masculino , Proteínas de Unión a la Región de Fijación a la Matriz/agonistas , Proteínas de Unión a la Región de Fijación a la Matriz/antagonistas & inhibidores , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Ratones , MicroARNs/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de Transcripción/agonistas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
Heat shock proteins have protective effects when cells are exposed to stress. However, the relationship between extracellular heat shock protein 70 (eHSP70) and osteogenesis of hMSCs has not been reported. The results of this study showed that HSP70 (200 ng/ml) increases alkaline phosphatase activity and promotes hMSC mineralization. Under osteogenic induction conditions, HSP70 significantly upregulated the expression of osteo-specific genes, such as the runt family transcription factor Runx2 and osterix (OSX). Comparative expression profiling by microarray and pathway analyses revealed that HSP70 promotes osteogenesis of hMSCs through activation of the ERK signaling pathway. HSP70 may be a potential therapeutic agent for the treatment of bone nonunion.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Sistema de Señalización de MAP Quinasas , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Regulación hacia Arriba , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Biomarcadores/metabolismo , Butadienos/farmacología , Calcificación Fisiológica/efectos de los fármacos , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Espacio Extracelular , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Nitrilos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Factor de Transcripción Sp7 , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Mechanical stimuli play crucial roles in bone remodeling and resorption. Osteopontin (OPN), a marker for osteoblasts, is important in cell communication and matrix mineralization, and is known to function during mechanotransduction. Hypergravity is a convenient approach to forge mechanical stimuli on cells. It has positive effects on certain markers of osteoblast maturation, making it a possible strategy for bone tissue engineering. We investigated the effects of hypergravity on OPN expression and cell signaling in osteoblasts. Hypergravity treatment at 20 g for 24 hours upregulated OPN expression in MC3T3-E1 cells at the protein as well as mRNA level. Hypergravity promoted OPN expression by facilitating focal adhesion assembly, strengthening actin bundles, and increasing Runx2 expression. In the hypergravity-triggered OPN expression pathway, focal adhesion assembly-associated FAK phosphorylation was upstream of actin bundle assembly.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Adhesiones Focales/metabolismo , Hipergravedad , Mecanotransducción Celular/genética , Osteoblastos/metabolismo , Osteopontina/genética , ARN Mensajero/genética , Actinas/genética , Actinas/metabolismo , Animales , Diferenciación Celular , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/agonistas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/ultraestructura , Regulación de la Expresión Génica , Humanos , Ratones , Osteoblastos/ultraestructura , Osteopontina/agonistas , Osteopontina/antagonistas & inhibidores , Osteopontina/metabolismo , Fosforilación , ARN Mensajero/agonistas , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Xanthohumol (XN), the principal prenylated flavonoid from the hop plant, an additive that contributes bitterness and flavor to beer, is known to be a potent phytoestrogen. Although XN has been identified as a chemopreventive agent and as an anti-infective agent, its effects on bone are unknown. In the present study, the effects of XN on osteoblast differentiation and function were determined by analyzing the activity of alkaline phosphatase (ALP), an osteoblast marker, and the regulation of RUNX2, a master gene of osteoblast differentiation, in a mesenchymal stem cell line. XN upregulated ALP activity and the expression of osteogenic marker genes. Additionally, XN increased the expression and transcriptional activity of RUNX2. To determine which signaling pathways are involved in the osteogenic effects of XN, we tested the effect of inhibitors of kinases known to regulate RUNX2. Enhancement of the transcriptional activity and expression of RUNX2 were inhibited by treatment with a p38 and an ERK inhibitor. These findings suggest that XN stimulates osteoblast differentiation by activation of RUNX2 via mechanisms related to the p38 MAPK and ERK signaling pathway. Regulation of RUNX2 activation by XN may be an important therapeutic target for osteoporosis.