RESUMEN
A growing body of research has attended to the experiences of transgender and gender non-conforming (TGN) youth's gender identity development. However, practical and ethical concerns have impeded our ability to understand the experiences of TGN youth. Thus, the aim of this study was to utilize one-on-one semi-structured interviews to explore White American TGN adults' (N = 15) retrospective accounts of their gender identity development in childhood and adolescence. Findings demonstrate considerable heterogeneity in TGN adults' retrospective accounts of their gender identity development. However, TGN adults consistently highlighted the role of social (e.g. friends, family and teachers) and contextual (e.g. online, offline, educational and geographical) factors in their gender identity journeys. This study provides new insight into the role of social and contextual factors in TGN adults' retrospective accounts of their gender identity development, demonstrating the importance of continuing to examine these factors in gender diversity research.
RESUMEN
The interaction among topological defects can induce novel phenomena such as disclination pairs in liquid crystals and superconducting vortex lattices. Nanoscale topological vortices with swirling ferroelectric, magnetic, and structural antiphase relationships were found in multiferroic h-YMnO(3). Herein, we report the discovery of intriguing, but seemingly irregular configurations of a zoo of topological vortices and antivortices. These configurations can be neatly analyzed in terms of graph theory and reflect the nature of self-organized criticality in complexity phenomena. External stimuli such as chemistry-driven or electric poling can induce the condensation and eventual annihilation of topological vortex-antivortex pairs.
Asunto(s)
Compuestos de Hierro/química , Electroquímica , Microscopía Electrónica de Transmisión/métodos , Estructura Molecular , Nanotecnología , Itrio/químicaRESUMEN
Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts, that plays a prominent role in degrading Type I collagen. Growing CatK null mice have osteopetrosis associated with a reduced ability to degrade bone matrix. Bone strength and histomorphometric endpoints in young adult CatK null mice aged more than 10 weeks have not been studied. The purpose of this paper is to describe bone mass, strength, resorption, and formation in young adult CatK null mice. In male and female wild-type (WT), heterozygous, and homozygous CatK null mice (total N=50) aged 19 weeks, in-life double fluorochrome labeling was performed. Right femurs and lumbar vertebral bodies 1-3 (LV) were evaluated by dual-energy X-ray absorptiometry (DXA) for bone mineral content (BMC) and bone mineral density (BMD). The trabecular region of the femur and the cortical region of the tibia were evaluated by histomorphometry. The left femur and sixth lumbar vertebral body were tested biomechanically. CatK (-/-) mice show higher BMD at the central and distal femur. Central femur ultimate load was positively influenced by genotype, and was positively correlated with both cortical area and BMC. Lumbar vertebral body ultimate load was also positively correlated to BMC. Genotype did not influence the relationship of ultimate load to BMC in either the central femur or vertebral body. CatK (-/-) mice had less lamellar cortical bone than WT mice. Higher bone volume, trabecular thickness, and trabecular number were observed at the distal femur in CatK (-/-) mice. Smaller marrow cavities were also present at the central femur of CatK (-/-) mice. CatK (-/-) mice exhibited greater trabecular mineralizing surface, associated with normal volume-based formation of trabecular bone. Adult CatK (-/-) mice have higher bone mass in both cortical and cancellous regions than WT mice. Though no direct measures of bone resorption rate were made, the higher cortical bone quantity is associated with a smaller marrow cavity and increased retention of non-lamellar bone, signs of decreased endocortical resorption. The relationship of bone strength to BMC does not differ with genotype, indicating the presence of bone tissue of normal quality in the absence of CatK.
Asunto(s)
Densidad Ósea/fisiología , Huesos/enzimología , Huesos/fisiología , Catepsinas/deficiencia , Osteogénesis , Animales , Fenómenos Biomecánicos , Resorción Ósea/enzimología , Huesos/anatomía & histología , Catepsina K , Catepsinas/metabolismo , Femenino , Fémur/anatomía & histología , Fémur/enzimología , Vértebras Lumbares/enzimología , Vértebras Lumbares/fisiología , Masculino , Ratones , Microscopía Fluorescente , Tamaño de los Órganos , Análisis de Regresión , Caracteres Sexuales , Propiedades de SuperficieRESUMEN
1. The disposition of 3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl) propyl]-imidazolidin-1-yl]-3(S)-(6-methoxy-pyridin-3-yl)propionic acid (compound A), a potent and selective alpha(v)beta(3) antagonist, was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 2. Compound A exhibited marked species differences in pharmacokinetics; the plasma clearances and bioavailabilities ranged from 33-47 ml min(-1) kg(-1) in rats and mice to 4-9 ml min(-1) kg(-1) in dogs and monkeys, and about 20% in rats to 70-80% in dogs and monkeys, respectively. Both the intravenous (i.v.) and oral kinetics of compound A were linear over the dose range studied in dogs (0.1-5 mg kg(-1) i.v. and 0.25-20 mg kg(-1) orally [p.o.]) and rats (1-30 mg kg(-1) i.v. and 4-160 mg kg(-1) p.o.). 3. Compound A was eliminated substantially by urinary excretion; the urinary recovery of the unchanged drug was 67% in rhesus, 48% in dogs and about 30% in rats. In these animal species, biotransformation was modest. 4. Following i.v. administration of [(14)C]-compound A to rats, the radioactivity rapidly distributed to all tissues investigated, with high levels of the radioactivity detected in liver, kidney and intestine soon after the drug administration. The radioactivity declined rapidly, with less than 1% of the i.v. dose remaining at 30-h post-dose. 5. Compound A was moderately bound to plasma proteins, with unbound fractions of 26, 20, 14 and 5% for rats, dogs, monkeys and humans, respectively. It was bound primarily to human alpha(1)-acid glycoprotein (about 85% binding at 0.1% concentration), as compared with human albumin (< 50% binding at 4% concentration). 6. Using simple allometry, compound A was predicted to exhibit relatively low clearance (1-3 ml min(-1) kg(-1)) and low volume of distribution (0.1-0.3 l kg(-1)) in humans. Based on the predicted values, compound A was projected to exhibit a favourable oral pharmacokinetic profile in humans, with good bioavailability (50-80%). These predicted values provided a basis for compound selection for further development.
Asunto(s)
Integrina alfaVbeta3/antagonistas & inhibidores , Naftiridinas/farmacocinética , Succinimidas/farmacocinética , Administración Oral , Animales , Proteínas Sanguíneas/metabolismo , Radioisótopos de Carbono , Perros , Evaluación Preclínica de Medicamentos , Femenino , Predicción , Humanos , Infusiones Intravenosas , Integrina alfaVbeta3/metabolismo , Macaca mulatta , Masculino , Tasa de Depuración Metabólica , Ratones , Naftiridinas/sangre , Naftiridinas/química , Naftiridinas/orina , Unión Proteica , Ratas , Ratas Sprague-Dawley , Succinimidas/sangre , Succinimidas/química , Succinimidas/orinaRESUMEN
Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.
Asunto(s)
Huesos/metabolismo , Dinoprostona/metabolismo , Receptores de Prostaglandina E/metabolismo , Animales , Huesos/efectos de los fármacos , Células Cultivadas , Humanos , Masculino , Periostio/citología , Ratas , Ratas Sprague-Dawley , Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E , Compuestos de Sulfhidrilo/farmacología , Tiofenos/farmacologíaRESUMEN
Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L with IC(50) values of 0. 37 and 0.45 M, respectively. Modification of compound 1 by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a K(i) value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chemical reactivity of the compound toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with human cathepsin K. The kinetic data obtained showed that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due to an increase of k(on) values and that the association of the compound with the enzyme fits an apparent one-step mechanism. (13)C NMR experiments performed with the enzyme papain showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by (13)C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC(50) value of 0.7 M. 1-Cyanopyrrolidine represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and proteolysis of bone collagen.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/síntesis química , Endopeptidasas , Nitrilos/síntesis química , Pirrolidinas/síntesis química , Sulfonamidas/síntesis química , Animales , Resorción Ósea/patología , Huesos/efectos de los fármacos , Huesos/metabolismo , Dominio Catalítico , Catepsina K , Catepsina L , Bovinos , Colágeno/metabolismo , Cisteína/química , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacocinética , Inhibidores de Cisteína Proteinasa/farmacología , Glutatión/química , Humanos , Técnicas In Vitro , Cinética , Espectroscopía de Resonancia Magnética , Nitrilos/química , Nitrilos/farmacocinética , Nitrilos/farmacología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Pirrolidinas/química , Pirrolidinas/farmacocinética , Pirrolidinas/farmacología , Conejos , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologíaRESUMEN
Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.
Asunto(s)
Naftiridinas/síntesis química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Propionatos/síntesis química , Sulfonamidas/síntesis química , Animales , Resorción Ósea/patología , Línea Celular , Técnicas de Cultivo , Humanos , Ligandos , Naftiridinas/química , Naftiridinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Propionatos/química , Propionatos/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.
Asunto(s)
Antígenos Transformadores de Poliomavirus , Antineoplásicos/farmacología , Osteocitos/citología , Osteocitos/efectos de los fármacos , Tretinoina/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno/genética , Medios de Cultivo Condicionados/farmacología , Sinergismo Farmacológico , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación Neoplásica de la Expresión Génica , Integrina alfa1beta1 , Integrinas/análisis , Integrinas/genética , Osteocitos/enzimología , ARN Mensajero/análisis , Ratas , Tibia/citologíaRESUMEN
Prostaglandin E stimulates bone formation in humans and animals, and increases intracellular cAMP in osteoblastic cells. We found that cAMP inhibits apoptosis in osteoblastic cells, and examined the mechanism of this effect. We report that the cAMP elevating agent, forskolin, increases cell number in the rat periosteal cell line (RP-11), by suppressing apoptosis in a cell type-specific manner. In RP-11, forskolin transiently up-regulates extracellular signal-regulated kinase activity, a known suppressor of apoptosis. PD98059, a selective inhibitor of the extracellular signal-regulated kinase pathway, only partially reverses the antiapoptotic effect of forskolin, which suggests an additional mechanism for cAMP action. We found that forskolin stimulates cytosolic sphingosine kinase (SPK) activity in these cells; in two other osteoblastic cell lines, however, forskolin does not suppress apoptosis. In contrast to the partial opposing effect of PD98059 to forskolin action, N, N-dimethylsphingosine, a specific inhibitor of SPK, completely reverses the antiapoptotic effect of forskolin, and has no effect on apoptosis in the absence of forskolin. These findings show for the first time that cAMP activates SPK in a cell-type-specific manner, and suggest that cAMP suppression of apoptosis in RP-11 periosteal cells is mediated by its stimulation of SPK.
Asunto(s)
Apoptosis/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/metabolismo , Periostio/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Recuento de Células/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Activación Enzimática , Periostio/citología , Periostio/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Integrins that bind RGD (arginine-glycine-aspartic acid) containing peptides, especially the vitronectin receptor alpha(v)beta3, have been implicated in the regulation of osteoclast function. Echistatin, an RGD-containing snake venom peptide with high affinity for beta3 integrins, as well as nonpeptide RGD mimetics, were shown to inhibit osteoclastic bone resorption in vitro and in vivo. To evaluate the role of RGD-binding integrins in bone metabolism, we examined by several methods the effects of echistatin on ovariectomy (OVX)-induced bone loss in mice and rats. First, we confirmed that echistatin binds in vitro with high affinity (Kd, 0.5 nM) to alpha(v)beta3 integrin purified from human placenta and established a competitive binding assay to measure echistatin concentrations in serum. We find that echistatin infused for 2 or 4 weeks at 0.36 microg/h x g body weight (approximately 50 nmol/day x mouse) completely prevents OVX-induced cancellous bone loss in the distal femora of ovariectomized mice. Echistatin has no effect on uterine weight, body weight, and femoral length changes induced by OVX, nor does it cause any apparent changes in major organs other than bone. In OVX rats, echistatin infusion at 0.26 microg/h x g for 4 weeks effectively prevents bone loss, evaluated by dual energy x-ray absorptiometry of the femur, by femoral ash weight, and by bone histomorphometry of the proximal tibia. At effective serum concentrations of 20-30 nM, measured at the end of the infusion period, echistatin maintains histomorphometric indices of bone turnover at control levels but does not decrease osteoclast surface. In conclusion, these results provide in vivo evidence, at the level of bone histology, that RGD-binding integrins, probably alpha(v)beta3, play a rate-limiting role in osteoclastic bone resorption and suggest a therapeutic potential for integrin ligands in the suppression of bone loss.
Asunto(s)
Resorción Ósea/prevención & control , Péptidos/farmacología , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Femenino , Péptidos y Proteínas de Señalización Intercelular , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Ovariectomía , Péptidos/sangre , Ratas , Ratas Sprague-Dawley , Receptores de Vitronectina/fisiologíaRESUMEN
Integrins are a large family of heteromeric cell surface receptors composed of non-covalently bound alpha and beta subunits which interact with extracellular matrix molecules, serum constituents and the adhesion molecules of the immunoglobulin family. The extracellular domains of many integrins recognize the RGD (Arg-Gly-Asp) tripeptide found in several extracellular macromolecules such as fibronectin, vitronectin, fibrinogen and osteopontin. The vitronectin receptor, alpha v beta 3 integrin, is highly expressed in osteoclasts, the bone resorbing cells, and binds many of these RGD containing proteins including osteopontin, which is abundant in bone. Antibodies to alpha v beta 3, RGD peptides and RGD containing proteins such as echistatin, and kistrin were shown to inhibit bone resorption in vitro and in vivo. The identity of the alpha v beta 3 natural ligand and its mode of action in bone are so far not known. In addition to the very high levels of alpha v beta 3, mammalian osteoclasts also express alpha 2 beta 1, a collagen/laminin receptor and alpha v beta 1, another vitronectin receptor. Signaling events that follow substrate recognition by osteoclasts are not well understood. RGD containing peptides and proteins modulate [Ca2+] transients in osteoclasts and phosphatidylinositol 3-kinase and pp60c-src are associated with alpha v beta 3 in these cells. alpha v and beta 3 genes were shown to be regulated by the calciotropic hormone 1,25(OH)2D3 and by a number of cytokines known to be modulators of bone metabolism. In summary, elucidation of the interactions of osteoclast integrins with components of bone matrix, may lead to further understanding of the mechanism of bone resorption.
Asunto(s)
Integrinas/fisiología , Osteoclastos/metabolismo , Animales , Resorción Ósea/metabolismo , Humanos , Modelos Biológicos , Receptores de Vitronectina/fisiología , Transducción de Señal/fisiologíaRESUMEN
Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts.
Asunto(s)
Osteoclastos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Androstadienos/farmacología , Animales , Células de la Médula Ósea/citología , Huesos/citología , Adhesión Celular , Células Cultivadas , Citoesqueleto/enzimología , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Ratones , Osteoclastos/ultraestructura , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores de Vitronectina/metabolismo , Wortmanina , Proteína de Unión al GTP rhoARESUMEN
Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.
Asunto(s)
Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Expresión Génica , Osteoclastos/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Alendronato , Secuencia de Aminoácidos , Animales , Arsenicales/farmacología , Células de la Médula Ósea , Resorción Ósea , Clonación Molecular , Técnicas de Cocultivo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Cráneo/citología , Vanadatos/farmacologíaRESUMEN
This study describes the isolation and characterization of highly enriched mammalian osteoclast precursors, released by the "disintegrin" echistatin, from an osteoclast formation culture. Incubation of a 6-day coculture of mouse bone marrow cells and mouse osteoblastic cells (MB1.8) with echistatin (30 nM), an RGD-containing snake venom, for 20 min yielded an 88-95% pure population of tartrate-resistant acid phosphatase-positive cells, 1.5 x 10(5) cells per 150 cm2 culture dish. These cells were mostly mononucleated and based on the following characteristics are considered to be prefusion osteoclasts (pOC cells): (i) presence of calcitonin (CT) receptors documented by 125I-sCT autoradiography and cAMP generation in response to salmon calcitonin; (ii) expression of mRNAs for alpha v beta 3 integrin, osteopontin, 92-kDa type IV collagenase (matrix metalloproteinase 9), carbonic anhydrase II, OC-2 (an "osteoclastic" cysteine proteinase), and protein tyrosine phosphatase epsilon; and (iii) high level expression of pp60c-src protein. The pOC cells resorb bone (form "pits" on bone slices) but only in the presence of osteoblastic MB1.8 cells and 1,25(OH)2D3. Resorption was inhibited by CT. In conclusion, we describe a rapid, reproducible procedure to isolate virtually pure mammalian prefusion osteoclasts, which should help in the study of osteoclast formation, composition, and function.
Asunto(s)
Células de la Médula Ósea , Separación Celular/métodos , Osteoclastos/citología , Animales , Células Cultivadas , Integrinas/biosíntesis , Péptidos y Proteínas de Señalización Intercelular , Ratones , Osteoclastos/metabolismo , Osteopontina , Péptidos/farmacología , Sialoglicoproteínas/biosíntesis , Venenos de Víboras/farmacologíaRESUMEN
Bone formation is linked closely to angiogenesis. Because prostaglandin E2 (PGE2) is a potent stimulator of bone formation, its effects were evaluated on vascular endothelial growth factor, a secreted endothelial cell-specific mitogen, and a potent angiogenic protein. Prostaglandin E2 increased vascular endothelial growth factor protein in conditioned media of osteoblastic RCT-3 cells within 3 hours. Prostaglandin E2 also increased the steady-state levels of vascular endothelial growth factor mRNA in a dose-dependent manner. The increased expression of vascular endothelial growth factor mRNA produced by PGE2 was rapid (maximal at 1 hour) and was enhanced by the protein synthesis inhibitor cycloheximide (5 micrograms/ml). The increase in vascular endothelial growth factor mRNA by PGE2 was inhibited strongly by pretreatment for 3 hours with dexamethasone (10(-7) M). Stimulation of vascular endothelial growth factor by PGE2 and its suppression by dexamethasone implicate the involvement of vascular endothelial growth factor in bone metabolism.
Asunto(s)
Factores de Crecimiento Endotelial/biosíntesis , Linfocinas/biosíntesis , Osteoblastos/metabolismo , Animales , Línea Celular , Cicloheximida/farmacología , Dexametasona/farmacología , Dinoprostona/farmacología , Factores de Crecimiento Endotelial/metabolismo , Factores de Crecimiento Endotelial/fisiología , Expresión Génica , Técnicas In Vitro , Linfocinas/metabolismo , Linfocinas/fisiología , ARN Mensajero/genética , Ratas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Prostaglandins of the E series have been shown to be effective inducers of bone formation in vivo. In this study, the effects of PGE2 were evaluated in vivo using subcutaneous administration (3 mg/kg/d for 25 days) to ovariectomized rats or local application in the marrow cavity of tibiae of rats using biodegradable implants (0.13, 1.4 and 32 microg released over 8 days). Systemic treatment of rats with PGE2 stimulated cancellous bone formation in the metaphysis of the proximal tibiae as well as endocortical bone formation and de novo trabecular bone formation in the marrow cavity. Local delivery of PGE2 increased cancellous bone volume in the secondary spongiosa and cortical thickness (at 32 microg). Comparisons of prostanoid effects in vitro, in a bone-derived cell line, showed that PGF2alpha was a better stimulator of DNA synthesis than PGE2. PGF2alpha increased the steady state levels of IGF-I receptor mRNA while PGE2 increased IGF-I expression. Although the mechanism of bone formation by PGE2 is not known at this time, it is clear that PGE2 has powerful local anabolic effects on bone formation in vivo possibly by mediating responses to signals such as changes in mechanical force.
Asunto(s)
Huesos/metabolismo , Dinoprostona/metabolismo , Osteogénesis/fisiología , Animales , Huesos/citología , Huesos/efectos de los fármacos , Línea Celular , ADN/biosíntesis , Dinoprost/metabolismo , Dinoprost/farmacología , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Factor I del Crecimiento Similar a la Insulina/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Osteogénesis/efectos de los fármacos , Ovariectomía , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptor IGF Tipo 1/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estrés Mecánico , Tibia/efectos de los fármacos , Tibia/crecimiento & desarrollo , Tibia/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
PGE1 and PGE2 are potent stimulators of bone formation. Osteogenesis is strongly dependent on angiogenesis. Vascular endothelial growth factor (VEFG), a secreted endothelial cell-specific mitogen, has been implicated in physiological and pathological angiogenesis. The aim of this study was to examine the possible role of VEGF in PG stimulation of bone formation. We found that in rat calvaria-derived osteoblast-enriched cells and in the osteoblastic RCT-3 cell line PGE2 and E1 increased VEGF mRNA and protein levels. The increased expression of VEGF mRNA produced by PGE2 was rapid (maximal at 1 h), transient (declined by 3 h), potentiated by cycloheximide, and abolished by actinomycin D. PGE2 had no effect on VEGF mRNA stability, suggesting transcriptional regulation of VEGF expression by PGF2. Rp-cAMP, a cAMP antagonist, suppressed VEGF mRNA induced by PGE2, indicating cAMP mediation. The upregulation of VEGF expression by PGE2 in the preosteoblastic RCT-1 cells was potentiated by treatment with retinoic acid, which induces the differentiation of these cells. The upregulation of VEGF mRNA by PGE2 was inhibited by dexamethasone treatment. In addition, Northern blot analysis showed that VEGF mRNA is expressed in adult rat tibia. In summary, we documented, for the first time, the expression of VEGF in osteoblasts and in bone tissue. Stimulation of VEGF expression by PGs and its suppression by glucocorticoids, which, respectively, stimulate and suppress bone formation, strongly implicate the involvement of VEGF in bone metabolism.
Asunto(s)
Alprostadil/farmacología , Dinoprostona/farmacología , Factores de Crecimiento Endotelial/biosíntesis , Linfocinas/biosíntesis , Osteoblastos/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , AMP Cíclico/fisiología , Cicloheximida/farmacología , Dactinomicina/farmacología , Dexametasona/farmacología , Factores de Crecimiento Endotelial/genética , Femenino , Linfocinas/genética , Datos de Secuencia Molecular , Osteoblastos/efectos de los fármacos , Embarazo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Tibia/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
This study examines the effect of leukemia inhibitory factor (LIF) on preosteoblastic rat calvaria (RCT-1) cells, which acquire osteoblastic properties when treated with retinoic acid (RA). LIF potentiated the increase in alkaline phosphatase (AP) activity produced by RA. The LIF effect was time and dose dependent (EC50, approximately 1 pM). The earliest effects on AP activity were detected at 48 h, and maximal effects were observed after 72 h. RA increased AP mRNA about 2-fold at 3 h and 6-fold at 6 and 12 h. LIF further increased AP mRNA to 18-fold at 12 h. After RA treatment AP mRNA returned to control levels at 24 h, but in the presence of LIF, AP mRNA remained elevated at 24 and 72 h of treatment. When given alone, LIF had no effect on either AP activity or mRNA levels. Tumor necrosis factor-alpha and 1,25-dihydroxyvitamin D3 also potentiated the RA induction of AP, and interleukin-6 had a small effect, whereas granulocyte macrophage colony-stimulating factor had no effect. LIF alone had a small inhibitory effect on type 1 collagen mRNA, but did not oppose the stimulatory effect of RA. Consistent with these biological actions, LIF receptors were demonstrated on these cells. [125I]LIF bound to RCT-1 cells at 0 C with an apparent dissociation constant of 20 pM, and it was found that these cells express an average of 300 receptors/cell. Scatchard analyses showed a single class of high affinity binding site. LIF was internalized with an endocytic rate constant for occupied receptors of 0.03 min-1, and the apparent equilibrium dissociation constant at 37 C was 358 pM. These findings suggest that osteoblast precursor cells are among the target cells of LIF.
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Fosfatasa Alcalina/metabolismo , Inhibidores de Crecimiento/metabolismo , Interleucina-6 , Linfocinas , Osteoblastos/metabolismo , Tretinoina/farmacología , Adenilil Ciclasas/metabolismo , Fosfatasa Alcalina/genética , Animales , Calcitriol/farmacología , Línea Celular , Colágeno/genética , Factores Estimulantes de Colonias/farmacología , Sinergismo Farmacológico , Inducción Enzimática/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Factor Inhibidor de Leucemia , Osteoblastos/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.
Asunto(s)
AMP Cíclico/biosíntesis , Dinoprostona/farmacología , Interleucina-1/metabolismo , Osteosarcoma/metabolismo , Receptores Inmunológicos/análisis , Adenilil Ciclasas/análisis , Resorción Ósea/inducido químicamente , Sinergismo Farmacológico , Humanos , Interleucina-1/farmacología , Radioisótopos de Yodo , Receptores de Interleucina-1 , Células Tumorales CultivadasRESUMEN
In rat osteosarcoma (ROS 17/2.8) cells, which express osteoblastic features in culture, basic fibroblast growth factor (bFGF) reduces the level of alkaline phosphatase, type I collagen, and osteocalcin mRNA and increases osteopontin mRNA, independent of growth stimulation. The fibroblast growth factor (FGF) effects are dose dependent (EC50 about 6 pM) and are detected 24 h after addition of the growth factor. bFGF also reduces parathyroid hormone-stimulatable adenylate cyclase and alkaline phosphatase activity in these cells. Concomitant treatment with pertussis toxin (20 ng/ml) opposes the FGF effects. Although cyclic AMP elevating agents mimic pertussis toxin action on some parameters, they produce opposite effects on others, indicating that antagonism between pertussis toxin and bFGF is not mediated by cyclic AMP. bFGF caused a small reduction in steady state NAD-dependent ADP-ribosylation and had no detectable effects on the steady-state levels of the Gi alpha (alpha subunit of the inhibitory G protein) 1, 2, and 3, visualized with specific antibodies in these cells. Although the site of interaction of pertussis toxin and FGF remains to be determined, the findings presented here suggest separate control of growth and differentiation by bFGF and show that pertussis toxin treatment can modulate differentiation in these cells, presumably via Gi proteins.