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
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
Maximizing peak bone mass is an important factor in osteoporosis prevention. Resistance exercise increases bone mass and strength, while nutritional supplements have beneficial effects on bone loss reduction. We have previously shown that the combined intake of sucrose and amino acids (AA), which is strongly insulinogenic, efficiently increased muscle protein synthesis. To investigate the effects of sugar and an AA solution immediately after resistance exercise, we compared insulinogenic sucrose and non-insulinogenic fructose combined with an AA solution with or without resistance exercise. Sucrose intake immediately after resistance exercise increased the trabecular bone mass and compressive maximum load compared with fructose+AA intake after exercise. Additionally, combined sucrose+AA and exercise increased trabecular bone formation and decreased bone resorption more than combined fructose and exercise. Serum insulin levels were greatly increased by sucrose+AA intake with exercise. In culture experiments, neither sugar+AA affected osteoblast and osteoclast differentiation. In a gene expression study, sucrose+AA intake after resistance exercise was shown to upregulate the Runx2 expression level and decrease RANKL/OPG ratio. These results suggest that the combined intake of sucrose and an AA solution immediately after resistance exercise exerts anabolic effects on bone by altering gene expression related to bone remodeling. Although translation of our bone remodeling findings from animal to human studies has been challenging, our findings suggest that exercise with sugar+AA intake may contribute to improved bone health.
Asunto(s)
Aminoácidos/administración & dosificación , Huesos/fisiología , Fructosa/administración & dosificación , Insulina/biosíntesis , Condicionamiento Físico Animal , Sacarosa/administración & dosificación , Células 3T3 , Absorciometría de Fotón , Animales , Secuencia de Bases , Densidad Ósea , Cartilla de ADN , Ratones , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho(-/-)mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization - according to von Kossa's staining method - and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho(-/-)metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho(-/-)mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho(-/-)mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho(-/-)mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.
Asunto(s)
Enfermedades Óseas Metabólicas/patología , Calcificación Fisiológica/genética , Glucuronidasa/deficiencia , Osteoblastos/ultraestructura , Animales , Enfermedades Óseas Metabólicas/metabolismo , Huesos/química , Calcio/análisis , Microanálisis por Sonda Electrónica , Fémur , Glucuronidasa/genética , Proteínas Klotho , Magnesio/análisis , Masculino , Ratones , Ratones Mutantes , Microscopía Electrónica de Transmisión , Modelos Animales , Fósforo/análisis , TibiaRESUMEN
Osteopontin (OPN) is a major non-collagenous bone matrix protein implicated in the regulation of cell function. Although OPN is rich in the cementum of the tooth, the significance of OPN in this tissue is not understood. Tooth root resorption is the most frequent complication of orthodontic tooth movement (TM). The objective of this study was to examine the pathophysiological role of OPN in cementum of the tooth root. For this purpose, the upper right first molar (M1) in OPN-deficient and wild-type (WT) mice was subjected to mechanical force via 10 gf NiTi coil spring while the left side molar was kept intact to serve as an internal control. Micro-CT section and the level of tartrate resistant acid phosphatase (TRAP)-positive cells on the tooth root surface defined as odontoclasts were quantified at the end of the force application. In WT mice, force application to the tooth caused appearance of odontoclasts around the mesial surface of the tooth root resulting in tooth root resorption. In contrast, OPN deficiency significantly suppressed the force-induced increase in the number of odontoclasts and suppressed root resorption. This force application also induced increase in the number of TRAP-positive cells in the alveolar bone on the pressure side defined as osteoclasts, while the levels of the increase in osteoclastic cell number in such alveolar bone were similar between the OPN-deficient and WT mice. These observations indicate that OPN deficiency suppresses specifically tooth root resorption in case of experimental force application.
Asunto(s)
Osteoclastos/citología , Osteopontina/deficiencia , Resorción Radicular/patología , Animales , Fenómenos Biomecánicos , Calcio/metabolismo , Recuento de Células , Ratones , Ratones Noqueados , Pruebas de Neutralización , Osteoclastos/efectos de los fármacos , Fósforo/metabolismo , Tomografía Computarizada por Rayos X , Diente/efectos de los fármacos , Factores de Necrosis Tumoral/farmacologíaRESUMEN
Klotho-deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho-deficient mice-comparing the findings with those from their wild-type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization.
Asunto(s)
Dentina/anatomía & histología , Dentinogénesis/fisiología , Glucuronidasa/fisiología , Incisivo/anatomía & histología , Animales , Apoptosis/fisiología , Calcio/metabolismo , Dentina/metabolismo , Microanálisis por Sonda Electrónica , Inmunohistoquímica , Incisivo/metabolismo , Incisivo/fisiología , Proteínas Klotho , Magnesio/metabolismo , Ratones , Ratones Noqueados , Fósforo/metabolismoRESUMEN
Rheumatoid arthritis is one of the most critical diseases that impair the quality of life of patients, but its pathogenesis has not yet been fully understood. Osteopontin (OPN) is an extracellular matrix protein containing Arg-Gly-Asp (RGD) sequence, which interacts with alpha(v)beta3 integrins, promotes cell attachment, and cell migration and is expressed in both synovial cells and chondrocytes in rheumatoid arthritis; however, its functional relationship to arthritis has not been known. Therefore, we investigated the roles of OPN in the pathogenesis of inflammatory process in a rheumatoid arthritis model induced by a mixture of anti-type II collagen mAbs and lipopolysaccharide (mAbs/LPS). mAbs/LPS injection induced OPN expression in synovia as well as cartilage, and this expression was associated with joint swelling, destruction of the surface structures of the joint based on scanning electron microscopy, and loss of toluidine blue-positive proteoglycan content in the articular cartilage in wild-type mice. In contrast, OPN deficiency prevented the mice from such surface destruction, loss of proteoglycan in the articular joint cartilage, and swelling of the joints even when the mice were subjected to mAbs/LPS injection. Furthermore, mAbs/LPS injection in wild-type mice enhanced the levels of CD31-positive vessels in synovia and terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive chondrocytes in the articular cartilage, whereas such angiogenesis as well as chondrocyte apoptosis was suppressed significantly in OPN-deficient mice. These results indicated that OPN plays a critical role in the destruction of joint cartilage in the rheumatoid arthritis model in mice via promotion of angiogenesis and induction of chondrocyte apoptosis.