RESUMEN
The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency-induced skeletal diseases.
Asunto(s)
Canales de Calcio/metabolismo , Magnesio/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Animales , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Resorción Ósea/patología , Señalización del Calcio , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Inositol/farmacología , Lisosomas/metabolismo , Deficiencia de Magnesio/tratamiento farmacológico , Deficiencia de Magnesio/metabolismo , Deficiencia de Magnesio/patología , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Células RAW 264.7 , Sodio/metabolismoRESUMEN
Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Huesos/citología , Movimiento Celular , Proteínas Oncogénicas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Resorción Ósea/metabolismo , Resorción Ósea/patología , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Técnicas de Silenciamiento del Gen , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones Noqueados , Proteínas Oncogénicas/deficiencia , Tamaño de los Órganos , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Radiografía , Cráneo/efectos de los fármacos , Cráneo/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3'-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3'-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis.
Asunto(s)
Resorción Ósea/fisiopatología , Regulación de la Expresión Génica/fisiología , Osteoporosis/fisiopatología , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Absorciometría de Fotón , Factores de Edad , Animales , Densidad Ósea , Cartilla de ADN/genética , Imagenología Tridimensional , Luciferasas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estabilidad del ARN/genética , ARN Interferente Pequeño/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microtomografía por Rayos XRESUMEN
CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1ß was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1ß promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.
Asunto(s)
Artritis Experimental/genética , Cartílago Articular/inmunología , Metaloproteinasa 3 de la Matriz/inmunología , Proteínas Asociadas a Matriz Nuclear/inmunología , Ligando RANK/inmunología , Factores de Transcripción/inmunología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/inmunología , Artritis Experimental/patología , Autoanticuerpos/biosíntesis , Resorción Ósea , Cartílago Articular/patología , Condrocitos/inmunología , Condrocitos/patología , Femenino , Regulación de la Expresión Génica , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/inmunología , Sueros Inmunes/administración & dosificación , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Articulaciones/inmunología , Articulaciones/patología , Masculino , Metaloproteinasa 3 de la Matriz/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/deficiencia , Proteínas Asociadas a Matriz Nuclear/genética , Regiones Promotoras Genéticas , Ligando RANK/genética , Índice de Severidad de la Enfermedad , Transducción de Señal , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Ift88 is an intraflagella transport protein, critical for the cilium, and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how Ift88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF-ß on the expression of Ift88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF-ß on gene expression. TGF-ß treatment suppresses the levels of Ift88 mRNA in a dose-dependent manner starting from as low as 0.5 ng/mL and reaching the nadir at around 2 ng/mL. TGF-ß treatment also suppresses the protein levels of Ift88. TGF-ß suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF-ß suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF-ß treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF-ß treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF-ß-induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF-ß-induced regulation of extracellular matrix protein expression. TGF-ß also suppresses Ift88 mRNA expression in primary culture of rib chondrocytes. These data indicate that TGF-ß regulates Ift88 gene expression at least in part via posttrascriptional manner.
Asunto(s)
Cilios/metabolismo , Osteoartritis/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Animales , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Cilios/patología , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Osteoartritis/metabolismo , Osteoartritis/patología , Fosforilación , Transducción de Señal , Factor de Crecimiento Transformador beta/administración & dosificación , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
ß adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of ß2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Osteoblastos/metabolismo , Receptores Adrenérgicos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
As the aged population is soaring, prevalence of osteoporosis is increasing. However, the molecular basis underlying the regulation of bone mass is still incompletely understood. Sympathetic tone acts via beta2 adrenergic receptors in bone and regulates the mass of bone which is the target organ of parathyroid hormone (PTH). However, whether beta2 adrenergic receptor is regulated by PTH in bone cells is not known. We therefore investigated the effects of PTH on beta2 adrenergic receptor gene expression in osteoblast-like MC3T3-E1 cells. PTH treatment immediately suppressed the expression levels of beta2 adrenergic receptor mRNA. This PTH effect was dose-dependent starting as low as 1 nM. PTH action on beta2 adrenergic receptor gene expression was inhibited by a transcriptional inhibitor, DRB, but not by a protein synthesis inhibitor, cycloheximide suggesting direct transcription control. Knockdown of beta2 adrenergic receptor promoted PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular basis for this phenomenon, knockdown of beta2 adrenergic receptor enhanced PTH-induced transcriptional activity of cyclic AMP response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors also enhanced forskolin-induced luciferase expression, revealing that adenylate cyclase activity is influenced by beta2 adrenergic receptor. As for phosphorylation of transcription factor, knockdown of beta2 adrenergic receptor enhanced PTH-induced phosphorylation of cyclic AMP response element binding protein (CREB). These data reveal that beta2 adrenergic receptor is one of the targets of PTH and acts as a suppressor of PTH action in osteoblasts.
Asunto(s)
Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Animales , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Parathyroid hormone (PTH), the major calcium-regulating hormone, and norepinephrine (NE), the principal neurotransmitter of sympathetic nerves, regulate bone remodeling by activating distinct cell-surface G protein-coupled receptors in osteoblasts: the parathyroid hormone type 1 receptor (PTHR) and the ß(2)-adrenergic receptor (ß(2)AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of ß(2)AR via the sympathetic nervous system decreases bone formation and increases bone resorption. Conversely, daily injection of PTH (1-34), a regimen known as intermittent (i)PTH treatment, increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis. Here, we show that iPTH has no osteoanabolic activity in mice lacking the ß(2)AR. ß(2)AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of ß(2)AR blocks expression of iPTH-target genes involved in bone formation and resorption that are regulated by the cAMP/PKA pathway. These data implicate an unexpected functional interaction between PTHR and ß(2)AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism.
Asunto(s)
Huesos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Absorciometría de Fotón , Anabolizantes/metabolismo , Anabolizantes/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Huesos/diagnóstico por imagen , Huesos/metabolismo , Femenino , Fémur/efectos de los fármacos , Fémur/metabolismo , Fluoresceínas , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Hormona Paratiroidea/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptores Adrenérgicos beta 2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microtomografía por Rayos XRESUMEN
Bone metabolism is maintained via balanced repetition of bone resorption by osteoclasts and bone formation by osteoblasts. Osteoblastic cells are capable of conducting self-renewal and differentiation that are basically associated with cell-cycle transition to enable cell specification and bone formation. Osteoblasts are also migrating to fill the resorption cavity curved by osteoclasts during bone remodeling to maintain homeostasis of bone mass whose imbalance leads to osteoporosis. However, technical difficulties have hampered the research on the dynamic relationship between cell cycle and migration in osteoblasts. In this report, we overcome these problems by introducing fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter system in calvarial osteoblastic cells and reveal that the cells in G1 as well as S/G2 /M phase are migrating. Furthermore, the osteoblastic cells in S/G2 /M phase migrate faster than those in G1 phase. Interestingly, parathyroid hormone (PTH) as an anabolic agent enhances migration velocity of the cells. Mechanical stress, another anabolic signal, also enhances migration velocity. In contrast, in the presence of both PTH and mechanical stress, the migration velocity returns to the base line levels revealing the interaction between the two anabolic stimuli in the regulation of cell migration. Importantly, PTH and mechanical stress also interact when they regulate the transition of cell cycle. These data demonstrate that osteoblastic migration is linked to cell cycle and it is under the control of mechanical and chemical stimuli that coordinate to regulate bone mass.
Asunto(s)
Técnicas Biosensibles , Remodelación Ósea , Ciclo Celular , Movimiento Celular , Rastreo Celular/métodos , Mecanotransducción Celular , Osteoblastos/metabolismo , Hormona Paratiroidea/metabolismo , Animales , Células Cultivadas , Genes Reporteros , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , Estrés Mecánico , Factores de TiempoRESUMEN
The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the ß2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the ß2-adrenergic receptor/cAMP signaling system.
Asunto(s)
Huesos/fisiología , Osteopontina/metabolismo , Sistema Nervioso Simpático/fisiología , Análisis de Varianza , Animales , Huesos/metabolismo , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Isoproterenol/farmacología , Ratones , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteopontina/deficiencia , Receptores Adrenérgicos beta 2/metabolismo , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Licorice (liquorice) is a common food additive and is used in Chinese medicine. Excess licorice intake can induce atrial fibrillation. Patients with atrial fibrillation possess constitutively activated G protein-gated inwardly rectifying K+ (GIRK) channels. Whether licorice affects GIRK channel activity is unknown. We aimed to clarify the effects of licorice ingredients on GIRK current and the mechanism of action. EXPERIMENTAL APPROACH: A major component of licorice, glycyrrhizic acid (GA), and its metabolite, 18ß-glycyrrhetinic acid (18ß-GA), were tested. We performed electrophysiological recordings in Xenopus oocytes to examine the effects of GA and 18ß-GA on various GIRK subunits (Kir 3.1-Kir 3.4), mutagenesis analyses to identify the crucial residues for drug action and motion analysis in cultured rat atrial myocytes to clarify effects of 18ß-GA on atrial functions. KEY RESULTS: GA inhibited Kir 3.1-containing channels, while 18ß-GA activated all Kir 3.x subunits. A pore helix residue Phe137 in Kir 3.1 was critical for GA-mediated inhibition, and the corresponding Ser148 in Kir 3.2 was critical for 18ß-GA-mediated activation. 18ß-GA activated GIRK channel in a Gßγ -independent manner, whereas phosphatidylinositol 4,5-bisphosphate (PIP2 ) was essential for activation. Glu236 located at the cytoplasmic pore of Kir 3.2 appeared to be important to interactions with 18ß-GA. In rat atrial myocytes, 18ß-GA suppressed spontaneous beating via activation of GIRK channels. CONCLUSION AND IMPLICATIONS: GA acts as a novel GIRK inhibitor, and 18ß-GA acts as a novel GIRK activator. 18ß-GA alters atrial function via activation of GIRK channels. This study elucidates the pharmacological activity of licorice ingredients and provides information for drug design.
Asunto(s)
Fibrilación Atrial , Ácido Glicirretínico/análogos & derivados , Glycyrrhiza , Humanos , Ratas , Animales , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Proteínas de Unión al GTP/metabolismoRESUMEN
Cigarette smoke has been recognized as a major risk factor for cardiovascular disease. However, its direct effects on rodent and human cardiomyocytes and its cellular mechanisms are not fully understood. In this study, we examined the direct effects of cigarette smoke extract (CSE) on contractile functions, intracellular Ca2+ dynamics, and mitochondrial function using cultured or freshly isolated rat ventricular myocytes and human induced pluripotent stem cell (iPS)-derived cardiomyocytes. In rat cardiomyocytes, CSE (≥0.1%) resulted in a time- and concentration-dependent cessation of spontaneous beating of cultured cardiomyocytes, eventually leading to cell death, which indicates direct toxicity. In addition, 1% CSE reduced contractile function of freshly isolated ventricular myocytes. Similar contractile dysfunction (declined spontaneous beating rate and contractility) was also observed in human iPS-derived cardiomyocytes. Regarding intracellular Ca2+ dynamics, 1% CSE increased the Ca2+ transient amplitude by greatly increasing systolic Ca2+ levels and slightly increasing diastolic Ca2+ levels. CSE also accelerated the decay of Ca2+ transients, and triggered spike-shaped Ca2+ transients in some cells. These results indicate that CSE causes abnormal Ca2+ dynamics in cardiomyocytes. Furthermore, CSE induced a cascade of mitochondrial dysfunctions, including increased mitochondrial reactive oxygen species, opening of mitochondrial permeability transition pore, reduction of mitochondrial membrane potential, and release of cytochrome c from mitochondria. These results suggest that CSE-induced contractile dysfunction and myocardial cell death is caused by abnormal Ca2+ dynamics and subsequent mitochondrial dysregulation, which would result in reduced bioenergetics and activation of cell death pathways.
Asunto(s)
Fumar Cigarrillos , Células Madre Pluripotentes Inducidas , Enfermedades Mitocondriales , Humanos , Ratas , Animales , Ratas Sprague-Dawley , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Calcio/metabolismo , Enfermedades Mitocondriales/metabolismo , Productos de TabacoRESUMEN
Osteoclast differentiation is one of the critical steps that control bone mass levels in osteoporosis, but the molecules involved in osteoclastogenesis are still incompletely understood. Here, we show that two-pore channel 2 (TPC2) is expressed in osteoclast precursor cells, and its knockdown (TPC2-KD) in these cells suppressed RANKL-induced key events including multinucleation, enhancement of tartrate-resistant acid phosphatase (TRAP) activities, and TRAP mRNA expression levels. With respect to intracellular signaling, TPC2-KD reduced the levels of the RANKL-induced dynamic waving of Ca(2+) in RAW cells. The search for the target of TPC2 identified that nuclear localization of NFATc1 is retarded in TPC2-KD cells. Finally, TPC2-KD suppressed osteoclastic pit formation in cultures. We conclude that TPC2 is a novel critical molecule for osteoclastogenesis.
Asunto(s)
Fosfatasa Ácida/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Diferenciación Celular/fisiología , Isoenzimas/metabolismo , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Fosfatasa Ácida/genética , Animales , Canales de Calcio/genética , Línea Celular , Técnicas de Silenciamiento del Gen , Isoenzimas/genética , Masculino , Ratones , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/citología , Ligando RANK/genética , Fosfatasa Ácida TartratorresistenteRESUMEN
Bone development is a dynamic process that requires cell motility and morphological adaptation under the control of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure, and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment. However, the role of Pfn1 in regulation of mesenchymal progenitor cells (MPCs) during skeletal development is incompletely understood. Here we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1-deficient mice exhibited an absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location-specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone. In vitro, Pfn1 deficiency induced retardation of osteoblastic cell migration. These observations revealed that Pfn1 is a critical molecule for the skeletal development, and this could be at least in part associated with the retardation of cell migration.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Profilinas/fisiología , Alelos , Animales , Huesos/metabolismo , Cartílago/metabolismo , Movimiento Celular , Citoesqueleto/metabolismo , Genotipo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Células 3T3 NIH , Osteoblastos/citología , Osteogénesis , Profilinas/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transfección , Microtomografía por Rayos X/métodosRESUMEN
Mechanical stress is an important signal to determine the levels of bone mass. Unloading-induced osteoporosis is a critical issue in bed-ridden patients and astronauts. Many molecules have been suggested to be involved in sensing mechanical stress in bone, though the mechanisms involved in this phenomenon are not fully understood. Nck1 is an adaptor protein known to mediate signaling from plasma membrane-activated receptors to cytosolic effectors regulating actin cytoskeleton remodeling. Nck1 has also been implicated in cellular responses to endoplasmic reticulum stress. In vitro, in case of cell stress the actin cytoskeleton is disrupted and in such cases Nck1 has been reported to enter the nucleus of the cells to mediate the nuclear actin polymerization. However, the role of Nck1 in vivo during the bone response to mechanical stimuli is unknown. The purpose of this study is to examine the role of Nck1 in unloading-induced bone loss in vivo. Sciatic and femoral nerve resection was conducted. Neurectomy-based unloading enhanced Nck1 gene expression in bone about twofold. Using the Nck1 deficient mice and control Nck1+/+, effects of neurectomy-based unloading on bone structure were examined. Unloading reduced bone volume in wild type mice by 30% whereas the levels in bone loss were exacerbated to 50% in Nck1 deficient mice due to neurectomy after 4 weeks. These data demonstrate that Nck1 gene deficiency accelerates the mechanical unloading-induced bone loss suggesting Nck1 to be a crucial molecule in mechanical stress mediated regulation in bone metabolism.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Resorción Ósea/etiología , Proteínas Oncogénicas/deficiencia , Células 3T3 , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Fenómenos Biomecánicos , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Núcleo Celular/metabolismo , Desnervación , Nervio Femoral/cirugía , Expresión Génica , Suspensión Trasera/efectos adversos , Suspensión Trasera/fisiología , Humanos , Locomoción , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular/etiología , Atrofia Muscular/fisiopatología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/fisiología , Osteoblastos/metabolismo , Osteoblastos/patología , Nervio Ciático/cirugíaRESUMEN
Tumor necrosis factor-alpha (TNF-α) is a major inflammatory cytokine that induces interleukin (IL)-8 production. Although some studies have reported the involvement of the p38 MAPK signaling pathway in TNF-α-induced IL-8 production, its specific regulatory mechanisms in gingival epithelial cells (GECs) are still poorly understood. In the present study, Ca9-22 cells were used as representative GECs to investigate the effect of p38 signaling on TNF-α-induced IL-8 production. We found that TNF-α enhanced IL-8 production in Ca9-22 cells by activating the p38 signaling pathway, and one of its isoforms, p38α, played a key role. P38α deletion markedly inhibited TNF-α-induced IL-8 expression in Ca9-22 cells, while p38α gene rescue could reverse this effect. Further studies revealed that TNF-α-induced IL-8 production was markedly reduced when the threonine 180 and tyrosine 182 p38α phosphorylation sites were targeted for mutagenesis to alanine and phenylalanine, respectively, suggesting their critical role in the process. In conclusion, p38α plays an important role in TNF-α-induced IL-8 production, providing a potential therapeutic target to prevent and treat periodontal disease.
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Encía , Interleucina-8 , Factor de Necrosis Tumoral alfa , Humanos , Interleucina-8/biosíntesis , Interleucina-8/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular , Encía/metabolismoRESUMEN
Tumor metastasis to bone is a serious pathological situation that causes severe pain, and deterioration in locomoter function. However, the mechanisms underlying tumor metastasis is still incompletely understood. CIZ/NMP4 is a nucleocytoplasmic shuttling protein and its roles in tumor cells have not been known. We, therefore, hypothesized the role of CIZ/NMP4 in B16 melanoma cells that metastasize to bone. CIZ/NMP4 is expressed in B16 cells. The CIZ/NMP4 expression levels are correlated to the metastatic activity in divergent types of melanoma cells. Overexpression of CIZ/NMP4 increased B16 cell migration in Trans-well assay. Conversely, siRNA-based knockdown of CIZ/NMP4 suppressed migratory activity of these cells. As RANKL promotes metastasis of tumor cells in bone, we tested its effect on CIZ in melanoma cells. RANKL treatment enhanced CIZ/NMP4 expression. This increase of CIZ by RANKL promoted migration. Conversely, we identified CIZ/NMP4 binding site in the promoter of RANKL. Furthermore, luciferase assay indicated that CIZ/NMP4 overexpression enhanced RANKL promoter activities, revealing a positive feedback loop of CIZ/NMP4 and RANKL in melanoma. These observations indicate that CIZ/NMP4 is critical regulator of metastasis of melanoma cells.
Asunto(s)
Movimiento Celular/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas Asociadas a Matriz Nuclear/biosíntesis , Ligando RANK/metabolismo , Factores de Transcripción/biosíntesis , Animales , Sitios de Unión , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Adhesión Celular/genética , Retroalimentación Fisiológica , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Regiones Promotoras Genéticas , Ligando RANK/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
OBJECTIVE: Regeneration of bone requires the combination of appropriate drugs and an appropriate delivery system to control cell behavior. However, the delivery of multiple drugs to heal bone is complicated by the availability of carriers. The aim of this study was to explore a new system for delivery of a selective EP4 receptor agonist (EP4A) in combination with low-dose bone morphogenetic protein 2 (BMP-2). METHODS: Combined delivery of EP4A and BMP-2 was carried out with a nanogel-based scaffold in the shape of a disc, to repair critical-size circle-shaped bone defects in calvariae that otherwise did not heal spontaneously. RESULTS: Combination treatment with EP4A and low-dose BMP-2 in nanogel efficiently activated bone cells to regenerate calvarial bone by forming both outer and inner cortical plates as well as bone marrow tissue to regenerate a structure similar to that of intact calvaria. EP4A enhanced low-dose BMP-2-induced cell differentiation and activation of transcription events in osteoblasts. CONCLUSION: These data indicate that combined delivery of EP4A and low-dose BMP-2 via nanogel-based hydrogel provides a new system for bone repair.
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Enfermedades Óseas/tratamiento farmacológico , Proteína Morfogenética Ósea 2/uso terapéutico , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Polietilenglicoles/uso terapéutico , Polietileneimina/uso terapéutico , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Fosfatasa Alcalina/sangre , Animales , Enfermedades Óseas/fisiopatología , Proteína Morfogenética Ósea 2/farmacología , Huesos/efectos de los fármacos , Huesos/fisiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Nanogeles , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Fosforilación , Polietilenglicoles/farmacología , Polietileneimina/farmacología , Regeneración/efectos de los fármacos , Regeneración/fisiología , Proteína Smad4/metabolismo , Andamios del TejidoRESUMEN
Schnurri (Shn)-2 is a transcriptional modulator of bone formation and bone resorption and its deficiency causes low turnover state with higher cancellous bone mass due to the defects in osteoclasts that exceeds the defects in osteoblasts in mice. We addressed whether such low turnover of bone remodeling in Shn2 deficiency may be modulated in the absence of estrogen that induces high turnover state in vivo. Ovariectomy reduced bone mass in wild type compared to sham operated control mice and such reduction in bone mass was also observed in Shn2 deficient mice. However, due to the high levels of basal bone mass in Shn2 deficient mice, the bone mass levels after ovariectomy were still comparable to sham operated wild-type mice. Analysis indicated that estrogen depletion increased bone resorption at similar levels in wild type and Shn2 deficient mice though the basal levels of osteoclast number was slightly lower in Shn2-deficient mice. In contrast, basal levels of bone marrow cell mineralization in cultures were low in Shn2-deficeint mice while estrogen depletion increased the mineralization levels to those that were comparable to sham wild type. This indicates that Shn2-deficient mice maintain bone mass at the levels comparable to wild-type sham mice even after ovariectomy-induced bone loss and this correlates with the high levels of mineralization activity in bone marrow cells after ovariectomy.
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Resorción Ósea/metabolismo , Proteínas de Unión al ADN/deficiencia , Ovariectomía , Animales , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Calcificación Fisiológica , Recuento de Células , Proteínas de Unión al ADN/metabolismo , Pruebas de Enzimas , Femenino , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Tamaño de los Órganos , Osteoclastos/metabolismo , Osteoclastos/patología , OsteogénesisRESUMEN
Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.