RESUMEN
INTRODUCTION: Cleidocranial dysplasia (CCD, MIM#119600), for which the responsible gene is RUNX2, is a genetic disorder characterized by hypoplasia or aplasia of the clavicles, patent fontanelles, and a short stature. Supernumerary teeth and delayed eruption and impaction of permanent teeth are frequently associated with CCD. Our previous study reported wide intrafamilial variation in supernumerary tooth formation associated with a mutation in the RUNT-domain of RUNX2, suggesting a low correlation between the genotype and supernumerary tooth formation. To further clarify this point, a more precise evaluation was performed. DESIGN: Gene mutational analysis of nine Japanese individuals with CCD was performed. Dental and skeletal characteristics were examined based on patient examinations and radiographs. RESULTS: Four different gene mutations, including one novel mutation in RUNX2 gene (NM_001024630), were identified. Among them, four individuals had the R225Q mutation, three siblings had the P224S mutation, and the other two individuals had different frame-shift mutations. Wide variations in supernumerary tooth formation were observed in individuals with identical gene mutations, and discordance was seen between monozygotic twins. Asymmetric supernumerary tooth formation was noted in five out of the nine individuals. CONCLUSION: Individuals with identical gene mutations showed a wide variation in the supernumerary tooth formation. Not only the genotype but also environmental factors and a complex system including epigenetics and copy number variation might regulate supernumerary tooth formation in CCD.
Asunto(s)
Displasia Cleidocraneal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Mutación/genética , Diente Supernumerario/genética , Adenina , Adolescente , Adulto , Arginina/genética , Niño , Variaciones en el Número de Copia de ADN/genética , Enfermedades en Gemelos/genética , Epigénesis Genética/genética , Femenino , Mutación del Sistema de Lectura/genética , Heterogeneidad Genética , Variación Genética/genética , Genotipo , Glutamina/genética , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis Insercional/genética , Mutación Missense/genética , Mutación Puntual/genética , Prolina/genética , Serina/genética , Timina , Gemelos Monocigóticos/genéticaRESUMEN
INTRODUCTION: Hypohidrotic ectodermal dysplasia is a genetic disorder characterized by diminished or a lack of sweating, congenital missing teeth, and sparse or absent hair. Three genes, EDA, EDAR, and EDARADD, all related to tumor necrosis factor signaling, have been reported as responsible genes for this disorder. Among them, the largest numbers of mutations have been identified in EDA, and only two mutations identified in EDARADD. MATERIALS AND METHODS: DNA analysis of EDA, EDAR, and EDARADD was performed on a Mongolian patient by polymerase chain reaction-direct sequencing. RESULTS: The 5-year-old Mongolian individual had no erupted deciduous or permanent teeth. A panoramic radiograph showed only one tooth in the right mandible. His hair and eyebrows were sparse, but he did not have a short stature. He showed diminished sweating. The nails of his fingers and toes were normal. Based on these conditions, he was diagnosed with hypohidrotic ectodermal dysplasia. There was no gene mutation of EDA or EDAR. A novel heterozygous variant (P121S; c.361C>T) was identified in the death domain of EDARADD (NM_080738). No other member of his family was affected, and this variant was not identified in his parents or maternal grandparents. CONCLUSION: This study reports an individual affected with hypohidrotic ectodermal dysplasia with a novel heterozygous P121S variant in the death domain of EDARADD.
Asunto(s)
Pueblo Asiatico/genética , Displasia Ectodérmica Hipohidrótica Autosómica Recesiva/genética , Proteína de Dominio de Muerte Asociada a Edar/genética , Anodoncia/etiología , Anodoncia/genética , Preescolar , Enfermedades en Gemelos , Displasia Ectodérmica Hipohidrótica Autosómica Recesiva/complicaciones , Humanos , Masculino , Linaje , Mutación Puntual , Polimorfismo de Nucleótido SimpleRESUMEN
In the present paper, the involvement of active oxygen species in bone resorption has been studied. In order to compare the production of active oxygen by mouse marrow culture cells, fluorescence due to peroxides reacted with 2,7-dichlorofluorescin was measured. After marrow cells were cultured with 1,25-(OH)2D3 for 8 days, there were tartrate resistant acid phosphatase positive multinucleated cells (TRACP(+)MNCs), TRACP positive mononucleated cells, macrophage-like cells and marrow derived stromal cells. Among these cells, TRACP(+) cells could produce almost the equivalent amount of peroxides as could the macrophage-like cells. In order to examine the role of active oxygen in bone metabolism, the amount of oxidative stress was altered during the culture period in the same marrow culture system. Catalase, a catabolic enzyme of hydrogen peroxide (H2O2), significantly suppressed the formation of TRACP(+)MNCs in a dose dependent manner. This suppression was limited in the early stage of the culture period and was reduced by the addition of exogenous H2O2 to culture. Moreover, when superoxide dismutase, a converting enzyme from superoxide anion to H2O2, was added in this system, the formation of TRACP(+)MNCs was significantly increased. These results strongly suggest that active oxygen species, especially H2O2, may be involved in the regulation of osteoclast formation.
Asunto(s)
Médula Ósea/metabolismo , Osteoclastos/metabolismo , Consumo de Oxígeno , Fosfatasa Ácida/metabolismo , Animales , Células de la Médula Ósea , Resorción Ósea/metabolismo , Catalasa/metabolismo , Diferenciación Celular , Femenino , Peróxido de Hidrógeno/metabolismo , Isoenzimas/metabolismo , Ratones , Osteoclastos/citología , Oxidación-Reducción , Fosfatasa Ácida TartratorresistenteRESUMEN
Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca+ channels, we expressed alpha2, beta, and gamma DHPR subunits and a chimeric DHPR alpha(i) subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca2+ transient (CaT) in the absence of extracellular Ca2+ ([Ca2+]o). However, in the presence of [Ca2+]o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca2+ concentration ([Ca2+]i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+]i which lasted for 5-10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ channels, whereas the second component resulted from Ca2+ release through the RyR expressed in the membrane of internal Ca2+ stores. However, the onset and the rate of Ca2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca2+ current. These results suggest that the skeletal muscle RyR isoform supports Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional "close coupling" like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Calcio/farmacología , Proteínas Musculares/metabolismo , Animales , Células CHO , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Canales de Calcio Tipo L , Cricetinae , Electrofisiología , Cinética , Potenciales de la Membrana/fisiología , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Técnicas de Placa-Clamp , Plásmidos , Canal Liberador de Calcio Receptor de Rianodina , Transfección/fisiologíaRESUMEN
The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.
Asunto(s)
Condrocitos/citología , Cóndilo Mandibular/citología , Proteínas/fisiología , Animales , Apoptosis , Cartílago Articular/química , Cartílago Articular/citología , División Celular , Condrocitos/química , Colágeno/análisis , Cóndilo Mandibular/química , Ratones , Ratones Noqueados , Hormona Paratiroidea/análisis , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Hormona Paratiroidea/análisis , Tibia/química , Tibia/citologíaRESUMEN
It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.
Asunto(s)
Desarrollo Óseo/fisiología , Proteínas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Anhidrasa Carbónica II/metabolismo , Catepsina K , Catepsinas/metabolismo , Células Cultivadas , Femenino , Expresión Génica , Masculino , Mandíbula/crecimiento & desarrollo , Mandíbula/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteoclastos/metabolismo , Osteoclastos/patología , Osteopontina , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Proteínas/farmacología , Proteínas/fisiología , Sialoglicoproteínas/genéticaRESUMEN
Combined patch-clamp and fura-2 measurements were performed to investigate the mechanism that terminates Ca2+ release in rat skeletal myoballs. When cells were intracellularly perfused with solution containing 1 mM free Mg2+, the caffeine (10 mM)-induced Ca2+ transient was abruptly terminated by membrane repolarization (-70 mV). With low intracellular Mg2+ (e.g. 50 microM) perfusion, however, repolarization failed to terminate the caffeine transient. The results show that intracellular Mg2+ is necessary for repolarization-induced closing of the Ca2+ release channel.
Asunto(s)
Calcio/metabolismo , Magnesio/metabolismo , Músculo Esquelético/metabolismo , Animales , Cafeína/farmacología , Canales de Calcio/metabolismo , Células Cultivadas , Potenciales de la Membrana , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
Osteoclasts are multinucleated cells with the specialized function of resorbing calcified tissues. These cells develop from hemopoietic cells of the monocyte-macrophage lineage with the support of osteoblasts/stromal cells. Tooth eruption is a vertical movement of teeth via creation of an eruption pathway in and through the alveolar bone. The precise cellular and molecular determinants of tooth eruption are not yet clear, and a cell culture system that can reproduce the activity of osteoclast formation during tooth eruption is expected to be a useful tool to clarify the mechanism of eruption pathway formation. To this end, mandibular bodies, including incisors and molars, were isolated from 9- to 11-day-old mice undergoing active tooth eruption. Primary cells were obtained from mandibular bodies by enzymatic digestion and cultured in alphaMEM containing 15% FBS without any cytokine or growth factor or hormone in the culture (AFT culture, for alveolar bone, dental follicle, and tooth). A progressive increase in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells was observed in AFT culture. The osteoclastic cells generated were immunopositive for cathepsin K and calcitonin receptor, and formed resorption pits when cultured on dentine slices. Parathyroid hormone-related protein (PTHrP), expressed by the enamel organ of tooth, is reported to be an essential factor in creation of the eruption pathway. To verify this point, cells were isolated from mandibular bodies from which all teeth and dental follicles had been removed and cultured similarly (A culture, for alveolar bone). Osteoclastic cells were not formed and PTHrP production was hardly detected in the medium of A culture, in contrast to the high level of PTHrP in AFT culture. Since our previous study demonstrated that neonatal homozygous PTHrP-knockout mice show impaired osteoclastogenesis around tooth germs, AFT culture was performed by using this sample to examine whether this culture system can reproduce the status of osteoclastogenesis observed in vivo. The result showed that none of the osteoclastic cells were generated from cells of homozygous mice. We here report a novel mouse osteoclast culture system that reproduces the activity of osteoclast formation around erupting teeth without addition of any cytokine or growth factor or hormone to the medium. Histological examination of various transgenic and mutant mice now offers valuable findings on studies of tooth eruption and the present culture system using these animals would be a powerful tool in clarifying the cellular and molecular mechanisms of eruption pathway formation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Incisivo/citología , Diente Molar/citología , Osteoclastos/citología , Animales , Humanos , Incisivo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Diente Molar/metabolismo , Osteoclastos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/deficiencia , Proteína Relacionada con la Hormona Paratiroidea/genética , Ratas , Ratas Sprague-Dawley , Diente no Erupcionado/anatomía & histología , Diente no Erupcionado/metabolismoRESUMEN
Parathyroid hormone-related protein (PTHrP) is involved in epithelial-mesenchymal cell interactions during development of various tissues and organs. Tooth germ development is a classical model for this interaction. In tooth germs, PTHrP is expressed in the enamel organ (epithelial component), whereas its major receptor, the type I PTH/PTHrP receptor is expressed in cells of the alveolar bone and dental follicle (mesenchymal components). To clarify the role of PTHrP during fetal tooth germ development, PTHrP gene-knockout mice were used for histochemical and ultrastructural analysis. In wild-type mice, osteoclastic cells were aligned predominantly in the inner aspects of the alveolar bone surrounding the developing tooth germs throughout the late embryonic (after embryonic, 17.5 days) and neonatal animals examined. In contrast, osteoblasts were predominant in corresponding areas of fetal homozygous PTHrP-gene knockout mice with only occasional osteoclasts. In such areas, cell-free surfaces showing cement line-like tartrate-resistant acid phosphatase (TRAP) reactions were frequently observed. In neonatal homozygous mice, bone spicules were often shown to penetrate and/or compress the enamel organ and caused partial destruction of the tooth germs. Osteoclasts were few in number in the inner aspects of the alveolar bone, and had poorly developed ruffled border. No morphological abnormality was noted in cells of the tooth germs proper. On bone surfaces away from developing tooth germs, functional osteoclasts with structural features similar to those in wild-type mice were observed in homozygous mice. These observations suggest that PTHrP is required to maintain an appropriate spatiotemporal arrangement of bone cells and osteoclast function, which are necessary for the normal development of tooth germ and alveolar bone encasing the tooth germ. The observation also demonstrates that PTHrP deficiency affects the structure and function of osteoclasts exclusively those located in the vicinity of the growing tooth germ.
Asunto(s)
Odontogénesis/genética , Odontogénesis/fisiología , Proteínas/genética , Proteínas/fisiología , Diente/crecimiento & desarrollo , Fosfatasa Ácida/metabolismo , Animales , Animales Recién Nacidos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Modelos Biológicos , Osteoclastos/citología , Osteoclastos/fisiología , Proteína Relacionada con la Hormona Paratiroidea , Germen Dentario/citología , Germen Dentario/embriología , Germen Dentario/crecimiento & desarrolloRESUMEN
Parathyroid hormone-related protein (PTHrP) is synthesized by a variety of tumors and is thought to be the main cause of the clinical syndrome of humoral hypercalcemia of malignancy (HHM). In addition to its parathyroid hormone (PTH)-like actions, novel actions of PTHrP on placental calcium transport and inhibition of in vitro osteoclast activity have been demonstrated. The fact that osteoblasts act as mediators of osteoclastic bone resorption prompted us to investigate whether nontranformed, osteoblastlike cells produce PTHrP. PTHrP has been detected in developing human fetal bones and in rat long bones in culture. For this study, osteogenic cells, CRP 5/4 and CRP 10/30, were employed. Both cell types represent clonal bone cell populations established from 1-day-old rats. While CRP 10/30 cells express the osteoblastic phenotype, CRP 5/4 cells resemble cells with preosteoblastic properties. With a radioimmunoassay (RIA), utilizing antiserum directed against the amino-terminal PTHrP(1-40), it was found that both cell types synthesize PTHrP constitutively. CRP 10/30 cells produce about twice as much as CRP 5/4 cells. Transforming growth factor-beta (TGF-beta 1) was shown to increase the synthesis of PTHrP in CRP 5/4 cells by about 2.5-fold, while in CRP 10/30 cells it caused an approximate 50% reduction of PTHrP. Employing the reverse transcriptase polymerase chain reaction (RT-PCR) technique it was found that both bone cell types express mRNA for PTHrP and that the modulation of the PTHrP mRNA levels by TGF-beta 1 in CRP 5/4, and to a lesser degree in CRP 10/30 cells, was reflected in a change in the level of PTHrP protein in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Osteoblastos/metabolismo , Biosíntesis de Proteínas , Animales , Autorradiografía , Secuencia de Bases , Resorción Ósea/fisiopatología , Células Cultivadas , Clonación Molecular , Medios de Cultivo Condicionados , Fibroblastos/citología , Fibroblastos/metabolismo , Hipercalcemia/etiología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Osteoclastos/citología , Proteína Relacionada con la Hormona Paratiroidea , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radioinmunoensayo , Ratas , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Our previous study showed that tooth germs at late embryonic stage [later than embryonic day 17.5 (E17.5)] and neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are compressed or penetrated by the surrounding alveolar bone tissue. In vivo and in vitro studies have shown that the development of the tooth germ proper is not disturbed, but insufficient alveolar bone resorption, due to the decreased number and hypofunction of osteoclasts, is the main cause of this abnormality. In addition to the insufficient alveolar bone resorption, progressive bone formation toward tooth germs was observed in homozygous mice, suggesting that accelerated bone formation also contributes to this abnormality. To further investigate this, homozygous mice at E14.0 and E15.5, when alveolar bone is forming, were used for histochemical and bone histomorphometric analyses. In contrast to the late embryonic stage, the alveolar bone did not yet compress developing tooth germs in homozygous mice on E14.0, but a larger amount of bone tissue was seen compared to wild-type littermates. Histomorphometric analysis of bone at E14.0 revealed that the osteoblast numbers and surfaces in the mandibles and in the bone collar of femora of homozygous mice were significantly higher than those of wild-type mice. However, unlike our previous study showing the osteoclast surface on E18.5 in homozygous mice to be significantly lower than that of wild-type mice, this study at E14.0 showed no significant difference between the two genotypes. To evaluate the amount of calcification around tooth germs, 3D images of mandibles were reconstructed from the calcein-labeled sections of the wild-type and mutant mice. Labeling was performed at E14.0, and the mice were sacrificed 1 h after the calcein injection to minimize the effect of bone resorption. Comparison of the 3D images revealed that the labeled surface was larger around developing tooth germs in homozygous mouse than in wild-type mouse. On day E15.5, osteoblasts approached the enamel organ of homozygous mice but this was not observed in wild-type mice. In this study, we report a systemic increase in osteoblast number and accelerated bone formation in homozygous PTHrP-knockout mice, both of which contribute to the abnormal tooth development.
Asunto(s)
Osteoblastos/patología , Osteogénesis/genética , Proteína Relacionada con la Hormona Paratiroidea/genética , Germen Dentario/patología , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Calcinosis/enzimología , Calcinosis/metabolismo , Calcinosis/patología , Condrocitos/patología , Fémur/química , Fémur/enzimología , Fémur/patología , Histocitoquímica , Isoenzimas/metabolismo , Mandíbula/química , Mandíbula/enzimología , Mandíbula/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/química , Osteoblastos/enzimología , Osteoclastos/enzimología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Fosfatasa Ácida Tartratorresistente , Germen Dentario/química , Germen Dentario/embriologíaRESUMEN
Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.
Asunto(s)
Núcleo Celular/metabolismo , Proteína SUMO-1/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Células COS , Factores de Transcripción COUP , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Biblioteca de Genes , Mutación , Regiones Promotoras Genéticas/genética , Receptores de Esteroides , Proteína SUMO-1/genética , Distribución Tisular , Factores de Transcripción , Técnicas del Sistema de Dos Híbridos , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity.
Asunto(s)
Antipirina/análogos & derivados , Endotelio Vascular/metabolismo , Depuradores de Radicales Libres , Animales , Antipirina/farmacología , Bovinos , Células Cultivadas , Edaravona , Endotelio Vascular/efectos de los fármacos , Leucocitos/metabolismo , Leucotrienos/toxicidad , Peróxidos Lipídicos/toxicidad , Mediciones Luminiscentes , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We investigated whether functional changes occur with incomplete cerebral ischemia which do not lead to neural cell death. If functional changes are recognized, it is necessary to clarify whether they occur immediately after ischemia or after a lag of a few days similar to the pathological changes. Long-term potentiation (LTP) in both the Schaffer collateral-CA1 and the perforant path-dentate gyrus synapses in halothane-anesthetized rats were examined 1 day and 4 days after 10 min clamping of the bilateral common carotid arteries. LTP was substantially attenuated after clamping of the bilateral common carotid arteries. In Schaffer-CA1 synapses, the inhibition of LTP was significant on both the 1 day and 4 days after-clamping group. In perforant path-dentate gyrus synapses, LTP was significantly inhibited on only the 4 days after-clamping group. These results suggest that functional damages may occur with incomplete ischemia without any histological damages. In the 1 day after-clamping group, LTP was reduced, but the changes in LTP differed from the inhibition of the 4 days after-clamping group. Therefore, a so-called delayed dysfunction might exist in the hippocampal neurons, despite absence of pathological changes.
Asunto(s)
Isquemia Encefálica/fisiopatología , Giro Dentado/fisiopatología , Potenciación a Largo Plazo/fisiología , Vía Perforante/fisiopatología , Potenciales de Acción/fisiología , Animales , Giro Dentado/irrigación sanguínea , Electrofisiología , Masculino , Vía Perforante/irrigación sanguínea , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The time-course effects of transient cerebral ischemia on nitric oxide (NO) formation in the rat hippocampus were evaluated by the consecutive determination of oxidative NO metabolites (NO2- and NO3-), using brain microdialysis under the freely moving condition. Bilateral carotid artery occlusion (CAO; 2-vessel occlusion, 2VO; 10 and 20 min) and combined vertebral artery occlusion (4VO; 10 min) produced a transient increase in hippocampal NO2- and NO3- levels, according to the duration and degree of ischemic insults. In addition, 4VO produced a gradual increase in hippocampal NO2- and NO3- levels over a 24 h period after reperfusion, which was abolished by an inducible NO synthase inhibitor, aminoguanidine (10 mg/kg, intraperitoneally). These findings suggest that the dynamic changes in oxidative NO metabolite levels reflect NO production following transient cerebral ischemia, which is possibly mediated in part by an inducible NO synthase, in the rat hippocampus.
Asunto(s)
Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Microdiálisis , Óxido Nítrico/biosíntesis , Animales , Isquemia Encefálica/fisiopatología , Soluciones para Diálisis/metabolismo , Hipocampo/irrigación sanguínea , Masculino , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismoRESUMEN
Runx2 (runt-related transcription factor 2) deficient mice lacked the mandibular condylar cartilage and the mandibular bone. The anlage of the condylar process consisted of mesenchymal condensation, which expressed Type I collagen mRNA and alkaline phosphatase activity, but not Type II collagen and aggrecan mRNAs. Therefore, the differentiation of the mandibular condylar cartilage stopped at the preosteoblast (skeletoblast) stage. The lateral pterygoid muscle was attached to this anlage, and relatively abundant mesenchymal condensations were also formed at the muscle-attaching sites, e.g. the anlage of the mandibular body, the angular and coronoid processes. Three-dimensional reconstruction models showed that each mesenchymal condensation was connected to one another, and roughly outlined the shape of the mandible. Meckel's cartilage in the Runx2-deficient mice had two ectopic cartilaginous processes to which the digastric and myohyoid muscles were attached. These findings indicate that Runx2 is essential for the formation of the mandibular condylar cartilage, as well as for normal development of Meckel's cartilage and that muscle tissues influence mandible morphology.
Asunto(s)
Cartílago/anomalías , Anomalías Craneofaciales/genética , Mandíbula/anomalías , Proteínas de Neoplasias/genética , Factores de Transcripción/genética , Fosfatasa Alcalina/metabolismo , Animales , Coristoma/genética , Colágeno Tipo I/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Mandíbula/patología , Músculos Masticadores/anomalías , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Modelos Anatómicos , Proteínas de Neoplasias/deficiencia , Osteoblastos/metabolismo , ARN Mensajero/genética , Factores de Transcripción/deficienciaRESUMEN
Previous studies using parathyroid hormone-related protein (PTHrP) null mutant mice have indicated severe abnormalities in the endochondral ossification, suggesting that PTHrP affects chondrocyte differentiation. In this study, we found in newborn PTHrP-deficient mice some deformities in the mandible that is formed via intramembranous ossification. The mandibular ramus was bent downwards and a prominent bone crest to which the deep layer of masseter muscle was tendinously attached was observed in the mandibular body. Transmission electron microscopic studies showed that active bone formation was progressing along the tendon fibers of the masseter muscle. The examination of 3-D reconstruction models indicated that the mandibular ramus was bent at the site of muscle attachment, which was shifted in the direction of the muscle fibers. Muscle fiber type analysis using myosin ATPase staining showed that the masseter muscle in the newborn PTHrP-deficient mice contained numerous type 2B fibers, demonstrating premature maturation of this muscle. Based on these findings, we speculated that premature maturation of the masseter muscle leads, probably due to increased tensile forces, to accelerated bone crest formation and subsequent bending of the mandibular ramus. These results further suggest that PTHrP is involved in the regulation of muscle development in normal animals.
Asunto(s)
Mandíbula/anomalías , Proteínas/fisiología , Animales , Edad Gestacional , Inmunohistoquímica , Músculo Masetero/química , Músculo Masetero/embriología , Músculo Masetero/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestructura , Miosinas/análisis , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/análisis , Proteínas/genética , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Hormona Paratiroidea/análisisRESUMEN
Among arachidonic acid metabolites, leukotriene B4 (LTB4) plays an important role in inflammation, such as in the activation, adhesion, chemotaxis, and invasion of leukocytes. In this paper, we examined the effect of LTB4 on endothelial cell injury induced by polymorphonuclear leukocytes (PMNLs). 51Cr release, a marker of cellular injury, was elicited from prelabeled endothelial cells when the cells were cocultured with PMNLs activated by phorbol ester (TPA, 12-O-tetradecanoyl-phorbol-13-acetate). Under this condition, pretreatment of PMNLs with LTB4 enhanced their injury in a dose-dependent manner (0.2-2 microM). However, LTB4 alone at any dose could not induce any cellular injury. We also determined the amount of active oxygen species produced by PMNLs in response to TPA. The intensity of luminol-dependent chemiluminescence, a marker of active oxygen production, in PMNLs was also increased by pretreatment with 1 microM LTB4. These data suggest that LTB4 enhances endothelial cell injury by the priming effect on active oxygen production in activated PMNLs.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Leucotrieno B4/farmacología , Neutrófilos/efectos de los fármacos , Oxígeno/fisiología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Humanos , Mediciones Luminiscentes , Acetato de TetradecanoilforbolRESUMEN
The mandibular condylar cartilage undergoes endochondral bone formation and is an important growth site in the mandible. Parathyroid hormone-related protein (PTHrP) has received attention as a physiological regulator attenuating chondrocytic differentiation and preventing apoptotic cell death. In order to examine the localization of PTHrP and its receptor during fetal development of the condylar cartilage, an immunohistochemical study of PTHrP and the type I PTH/PTHrP receptor was carried out. At day 15 of gestation, the condylar cartilage was evident and some chondrocytes showed positive staining for PTHrP. At day 16, the cartilage was increasing in length and width, and PTHrP was localized in the flattened and hypertrophic cell layers. After day 17, when endochondral bone formation had already started, PTHrP was mainly observed in the flattened cell layer and in a few layers of the hypertrophic chondrocytes. The localization of the type I PTH/PTHrP receptor was similar to that of PTHrP on days 15 and 16, and was broadly distributed at day 18. Apoptotic chondrocytes were scarcely observed on days 15 and 16, and only a few cells were present in the erosion front at day 18. This temporal and spatial localization of PTHrP and the type I PTH/PTHrP receptor suggests that PTHrP is a possible autocrine/ paracrine factor regulating condylar chondrocytic differentiation during development.
Asunto(s)
Cartílago Articular/metabolismo , Cóndilo Mandibular/metabolismo , Hormona Paratiroidea/metabolismo , Proteínas/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Animales , Apoptosis/fisiología , Cartílago Articular/embriología , Feto/metabolismo , Técnicas para Inmunoenzimas , Inmunohistoquímica , Cóndilo Mandibular/embriología , Ratones , Ratones Endogámicos C57BL , Proteína Relacionada con la Hormona Paratiroidea , Receptor de Hormona Paratiroídea Tipo 1 , Coloración y Etiquetado/métodosRESUMEN
In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossification in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.