RESUMEN
Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Osteoblastos/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Secuencia de Bases , Biomarcadores , Proteínas Portadoras/metabolismo , Células Cultivadas , Clonación Molecular , Dexametasona/farmacología , Regulación hacia Abajo/genética , Histocitoquímica , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transportadores de UreaRESUMEN
In Drosophila melanogaster, the role of the metallodisintegrin, Kuzbanian (kuz), is thought to involve activation of the Drosophila Notch receptor that plays a role in cell-fate determination during neurogenesis and myoblast differentiation. To understand the possible function(s) of a-disintegrin and metalloproteinase (ADAM10), the mammalian ortholog of kuz, in the skeleton, we studied its expression as well as the messenger RNA (mRNA) encoding one candidate substrate, the mammalian Notch2 receptor in bone, bone cells, and cartilage. In sections of neonatal rat tibiae, ADAM10 is expressed in specific regions of articular cartilage and metaphyseal bone. Expression of ADAM10 in articular cartilage occurs predominantly in superficial chondrocytes and becomes more sporadic with increasing distance from the articular surface. In bone, ADAM10 is expressed by periosteal cells, osteoblasts, and osteocytes at locations of active bone formation. Osteoclasts did not express ADAM10. Notch2 mRNA expression was not detectable in superficial chondrocytes. However it colocalized at all sites of ADAM10 expression in bone cells. In vitro, both primary human osteoblasts and osteoblast cell lines expressed a single 4.5 kb and 7.5 kb transcript of ADAM10 and the Notch2 receptor homolog, respectively. Subcellular localization of the ADAM10 protein in MG-63 cells was determined using immunofluorescent techniques. These observations showed clearly that the ADAM10 protein was expressed in the trans-Golgi network and on the plasma membrane. Western blot analysis of fractionated cells showed that, in the plasma membrane fraction, the previously characterized 58 kDa and 56 kDa isoforms were present, whereas, in the trans-Golgi network, the ADAM10 protein was present in several additional bands, possibly indicative of further interdomain processing of the ADAM10 protein. The metallodisintegrins (ADAMs) have several putative functions, including modulation of cell adhesion, membrane-associated proteolysis, and cell-cell signaling. These observations suggest that, in bone but not cartilage, ADAM10 has catalytic activity within the transGolgi network and may play a role in the activation of Notch receptor homologs. This implicates ADAM10 in cell-fate determination of osteoblast progenitor cells, possibly during skeletal development and normal bone remodeling. Plasma-membrane-associated ADAM10 may confer alternative functions.
Asunto(s)
Huesos/química , Desintegrinas/análisis , Proteínas de la Membrana/análisis , Metaloendopeptidasas/análisis , Receptores de Superficie Celular/análisis , Secuencia de Aminoácidos , Animales , Western Blotting , Catálisis , Línea Celular , Separación Celular , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Osteoblastos/química , Ratas , Receptores Notch , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares/químicaRESUMEN
Better understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.
Asunto(s)
Células de la Médula Ósea/metabolismo , Megacariocitos/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animales , Células Cultivadas , Células Clonales , Sangre Fetal/citología , Citometría de Flujo , Humanos , Inmunohistoquímica , Recién Nacido , Megacariocitos/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Signaling between the various types of cells found in bone is responsible for controlling the activity of osteoblasts and osteoclasts, and therefore the regulation of bone mass. Our identification of a neuronal glutamate transporter in osteoblasts and osteocytes suggests the possibility that bone cells may use the excitatory amino acid glutamate as a signaling molecule. In these studies we report the expression of different subtypes of glutamate receptors in osteoblasts and osteoclasts in vitro and in vivo. We have identified expression in human and rat bone cells of N-methyl-D-aspartate receptor-1 (NMDAR-1) and 2D subunits and PSD-95, the NMDA receptor clustering protein associated with signaling in the central nervous system. In situ hybridization and immunohistochemistry localized NMDAR-1 expression to osteoblasts and osteoclasts in human tissue sections. These findings strengthen the suggestion that glutamate is involved in signaling between bone cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácido Glutámico/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Receptores de N-Metil-D-Aspartato/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Sistema de Transporte de Aminoácidos X-AG , Animales , Composición de Base , Transporte Biológico , Homólogo 4 de la Proteína Discs Large , Ácido Glutámico/genética , Guanilato-Quinasas , Humanos , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/genética , Transducción de Señal/genéticaRESUMEN
The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by increased marrow adipose tissue formation. Reversal of this process may provide a novel therapeutic approach for osteopenic disorders. We have shown that cells cultured from human trabecular bone are not only osteogenic, but are able also to undergo adipocyte differentiation under defined culture conditions. Osteoblast differentiation was induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and adipocyte differentiation by dexamethasone (dex) plus 3-isobutyl-1-methylxanthine (IBMX) treatment. Adipogenesis was characterized by lineage-specific enzyme and gene activities, alpha-glycerophosphate-3-dehydrogenase activity, fatty acid binding protein, aP2 and lipoprotein lipase expression. Osteoblastogenesis was assessed by osteoblast characteristic 1,25(OH)2D3 induction of alkaline phosphatase activity and osteoblast-specific 1,25(OH)2D3-induced osteocalcin synthesis and release. We provide evidence for a common pluripotent mesenchymal stem cell that is able either to undergo adipogenesis or osteoblastogenesis, using clonal cell lines derived from human trabecular bone cell cultures. Adipogenesis can be induced also by long chain fatty acids and the thiazolidinedione troglitazone. Dex plus IBMX-induced adipogenesis can be inhibited by interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. Interestingly, and in contrast to extramedullary adipocyte differentiation as shown by mouse 3T3L-1 and a human liposarcoma SW872 cell line, trabecular bone adipogenesis was unaffected by insulin. Also, the formation of fully differentiated adipocytes from trabecular bone cells after troglitazone treatment and long chain fatty acids was dependent on increased expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma2 caused by dex plus IBMX. Specific inhibition of marrow adipogenesis and promotion of osteoblastogenesis of a common precursor cell may provide a novel therapeutic approach to the treatment of osteopenic disorders.
Asunto(s)
Adipocitos/efectos de los fármacos , Enfermedades Óseas Metabólicas/patología , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Osteoblastos/efectos de los fármacos , Tiazolidinedionas , Proteínas Supresoras de Tumor , 1-Metil-3-Isobutilxantina/farmacología , Adipocitos/metabolismo , Fosfatasa Alcalina/biosíntesis , Animales , Apolipoproteínas/biosíntesis , Calcitriol/farmacología , Proteínas Portadoras/biosíntesis , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Cromanos/farmacología , Citocinas/farmacología , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Glucocorticoides/farmacología , Humanos , Hipoglucemiantes/farmacología , Lipoproteína Lipasa/biosíntesis , Ratones , Proteína P2 de Mielina/biosíntesis , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Inhibidores de Fosfodiesterasa/farmacología , Tiazoles/farmacología , TroglitazonaRESUMEN
Without habitual exercise, bone is lost from the skeleton. Interactions between the effects of loading of bone and other osteotropic influences are thought to regulate bone mass. In an attempt to identify potential targets for therapeutic manipulation of bone mass, we used differential RNA display to investigate early changes in osteocyte gene expression following mechanical loading of rat bone in vivo. One gene found to be down-regulated by loading had high homology to a glutamate/ aspartate transporter (GLAST) previously identified only the mammalian CNS. RT-PCR analysis using primers targeted to the coding region of the published GLAST sequence amplified identical products from bone and brain (but not a range of other tissues). The amplicons were sequenced and found to be identical to the published CNS GLAST sequence. Northern analysis confirmed expression of GLAST mRNA in bone and brain, but not other tissues. In situ hybridization localized GLAST mRNA expression in rat bone to osteoblasts and osteocytes. A GLAST antibody localized high levels of protein expression to osteoblasts, and newly incorporated osteocytes. Interestingly, older osteocytes also expressed detectable levels of GLAST. Another neural glutamate transporter, GLT-1 was immunolocalized to the pericellular region of mononuclear bone marrow cells, while a further antibody to the EAAC-1 transporter failed to bind to bone cells. Five days after loading, GLAST protein expression was undetectable in osteocytes of loaded bone but present in control nonloaded sections, confirming the downregulation detected by differential display. On quiescent periosteal surfaces, GLAST expression was almost absent, while on surfaces where loading had induced cellular proliferation and bone formation, GLAST protein expression was elevated. In the CNS, the expression of glutamate transporters on neuronal membranes is associated with reuptake of released neurotransmitters at synapses, where they have a role in the termination of transmitter action. In this study, we describe for the first time, the expression of GLAST (and GLT-1) in bone, raising the possibility that excitatory amino acids may have a role in paracrine intercellular communication in bone. Manipulation of bone cell function by moderators of glutamate action could therefore provide novel treatments for bone diseases such as osteoporosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Osteocitos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Secuencia de Bases , Northern Blotting , Regulación de la Expresión Génica , Ácido Glutámico/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Osteocitos/citología , Reacción en Cadena de la Polimerasa , Ratas , Soporte de PesoRESUMEN
Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.
Asunto(s)
Citrato (si)-Sintasa/genética , Escherichia coli/enzimología , Genes Bacterianos , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Western Blotting , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/aislamiento & purificación , Citrato (si)-Sintasa/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , OligodesoxirribonucleótidosRESUMEN
1. Studies of carbachol-induced contractions on mini-pig bladder tissue strips in vitro demonstrated that antagonist drugs produced a rank order of potency similar to that observed in guinea-pig tissues: propantheline approximately atropine greater than oxybutynin greater than dicyclomine greater than HHSiD greater than imipramine greater than terodiline approximately AF-DX 116. The drugs appeared to show competitive antagonism and the tissues exhibited resistance to complete cholinergic blockade. 2. Cytometry performed in vivo on awake mini-pigs also showed that i.v. cholinergic antagonists produced a dose-dependent depression of peak intravesical bladder pressure (PvesP) during slow filling of the bladder using urethral catheters, with a rank order of potency: atropine greater than oxybutynin approximately propantheline greater than HHSiD approximately dicyclomine greater than terodiline. Other parameters of the cystometrogram were unaffected by the antagonists, except for residual volume, which generally increased after drug treatment. 3. Hexahydrosiladifenidol (HHSiD), an ileal-selective competitive muscarinic antagonist, was about as effective an antagonist as the clinically useful drugs oxybutynin or dicyclomine, both in vitro and in vivo, suggesting that HHSiD may have useful therapeutic effects for the treatment of urinary incontinence. 4. Correlation of the rank order of potency for muscarinic antagonism between mini-pigs and guinea-pigs was very high in vitro (r = 0.97, P less than 0.05), as was the correlation among the drugs for their ability to depress PvesP of the cystometrogram in vivo (r = 0.89, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Parasimpatolíticos/farmacología , Vejiga Urinaria/anatomía & histología , Incontinencia Urinaria/tratamiento farmacológico , Animales , Carbacol/farmacología , Femenino , Cobayas , Contracción Muscular/efectos de los fármacos , Porcinos , Porcinos Enanos , Vejiga Urinaria/efectos de los fármacosRESUMEN
Building codes and standards are enforceable by law, but are recognized as a minimum design basis relative to fire safety. The same system that mandates these codes does not, in a court of law, judge solely by the codes. In civil litigation involving death and injury from a building fire, the jury must evaluate the design based not only on the codes but also on what is reasonable. This paper will examine the civil trial and its impact on engineers, architects involved in building design, and manufacturers who supply materials and products for such buildings. Actual case histories will be cited and recommendations for a greater information feedback from trials to practicing designers and manufacturers will be presented.