RESUMEN
BACKGROUND: Auditory processing (AP) is commonly regarded as the perceptual processing of auditory information in the central nervous system. However, the degree to which higher level cognitive processes are involved in AP or its disorders is contentious. Furthermore, there is little evidence regarding the effects of nonauditory cognitive processes on the various tests of AP in common clinical usage and thus on clinical diagnoses of auditory processing disorder. PURPOSE: To determine the effects of increased cognitive demand, generated by using a dual-task paradigm, on performance on different AP tests and types of AP tests in common clinical usage. In addition, to investigate the relationship between executive function and changes in AP test performance associated with increased cognitive demand. RESEARCH DESIGN: Counterbalanced repeated measures design, with assessment of AP test performance both on its own and in a dual-task paradigm designed to increase cognitive demand. STUDY SAMPLE: Twenty-nine young adults, with no reported hearing, learning, language or attention difficulties, English as first language, and hearing and middle-ear status within normal limits. DATA COLLECTION AND ANALYSIS: Testing was completed within a single 90-min session. A selection of standard AP tests, representing both adaptive and nonadaptive tests, as well as tests employing difference scores, was administered. These were Competing Sentences Test, Dichotic Digits Test, Frequency Pattern Test (nonadaptive tests); and Listening in Spatialized Noise-Sentences test, conditions "same-voice, 0°", "different-voice, 0°", and "same-voice, 90°" (adaptive tests), from which the difference scores "talker advantage" and "spatial advantage" were also derived. Each AP test was completed on its own (alone condition), and simultaneously with a visually presented task (dual-task condition). Executive function was assessed using the phonemic subtest of the Verbal Fluency Test. Nonparametric statistical test procedures were used. RESULTS: All five AP measures obtained from the nonadaptive tests showed a significant performance decrement in the dual-task condition compared with the alone condition, with one exception because of a strong ceiling effect. By contrast, none of the three AP measures obtained from the adaptive tests showed a significant performance decrement in the dual-task condition. Furthermore, neither of the two AP measures based on difference scores showed a significant performance decrement, but this finding simply reflects the lack of significant decrements in the relevant raw scores. Consistent with past reports of associations between executive function and AP performance, a significant positive correlation was found between executive function scores and performance on the Dichotic Digits Test. However, there were no significant correlations between executive function scores and changes in AP test scores between alone and dual-task conditions. CONCLUSIONS: Performance on commonly used nonadaptive tests of AP was significantly compromised by the increased cognitive demand resulting from the dual-task paradigm. By contrast, performance on AP measures obtained by adaptive test procedures was not significantly affected. Further investigation of the resilience to increased cognitive demand of the adaptive tests used here, and other adaptive tests of AP, is warranted. Results from this study support the further development of computerized adaptive tests of AP for use in clinical test batteries.
Asunto(s)
Percepción Auditiva/fisiología , Cognición/fisiología , Adolescente , Adulto , Femenino , Pruebas Auditivas , Humanos , Masculino , Análisis y Desempeño de Tareas , Adulto JovenRESUMEN
Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.
Asunto(s)
Calcio/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteocitos/efectos de los fármacos , ARN Mensajero/genética , Proteínas Adaptadoras Transductoras de Señales , Biomarcadores/metabolismo , Proteínas Morfogenéticas Óseas/agonistas , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Calcio/metabolismo , Diferenciación Celular , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/agonistas , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteocitos/citología , Osteocitos/metabolismo , Osteoprotegerina/agonistas , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Endopeptidasa Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultivo Primario de Células , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose- and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Calcificación Fisiológica , Diferenciación Celular , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoblastos/citología , Osteocitos/citología , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Neutralizantes/farmacología , Proteínas Morfogenéticas Óseas/farmacología , Calcificación Fisiológica/efectos de los fármacos , Calcificación Fisiológica/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Humanos , Hipofosfatemia/metabolismo , Hipofosfatemia/patología , Péptidos y Proteínas de Señalización Intercelular , Ratones , Modelos Biológicos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocitos/efectos de los fármacos , Osteocitos/metabolismo , Endopeptidasa Neutra Reguladora de Fosfato PHEX/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that gamma-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-kappaB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/metabolismo , Osteoblastos/citología , Osteocitos/citología , Vitamina K/farmacología , Ácido 1-Carboxiglutámico/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Metabolismo/efectos de los fármacos , Fenotipo , Vitamina K 1/farmacología , Vitamina K 2/farmacología , Vitamina K 3/farmacología , Warfarina/farmacologíaRESUMEN
Polyethylene (PE) wear particles are associated with the osteolysis seen in aseptic loosening that leads to orthopaedic implant failure. While cells of the monocyte/macrophage lineage are implicated, evidence is now emerging that osteoblastic cells may also be affected by PE. In this study we investigated the effect of PE particles on osteoblasts, using a novel in vitro cell culture system that was developed to juxtapose cells and PE particles, replicating the 3-dimensional (3D) environment near implants. This system allowed normal human bone-derived cells (NHBC) to undergo differentiation into a mature osteocyte-like phenotype over a 21-28-day culture period. PE particles induced an increase in mRNA expression of the osteocyte markers E11, DMP-1 and SOST/sclerostin. NHBC responded to PE particles by increasing the mRNA expression of several genes associated with osteoclast formation and activity (RANKL, IL-8 and M-CSF) and decreased the expression of the osteoclast antagonist, OPG. PE also appeared to induce a switch in the RUNX2 control of gene expression from that of promoting matrix production (type I collagen) to inducing the expression of pro-osteoclastogenic genes. These results suggest that PE particles switch mature osteoblastic cells from an anabolic to a more catabolic phenotype. This concept was further supported by the finding that PE-induced expression of RANKL mRNA in the mouse osteocyte cell line, MLO-Y4. Overall, our results suggest that PE particles directly induce a change in the phenotype of mature osteoblasts and osteocytes, consistent with the net loss of bone near orthopaedic implants.
Asunto(s)
Técnicas de Cultivo de Célula , Osteoblastos/fisiología , Osteocitos/fisiología , Fenotipo , Polietilenos/metabolismo , Animales , Materiales Biocompatibles/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Humanos , Ensayo de Materiales , Ratones , Osteoblastos/citología , Osteocitos/citología , Tamaño de la Partícula , Falla de Prótesis , Ligando RANK/genética , Ligando RANK/metabolismoRESUMEN
We have recently shown that TNF-related weak inducer of apoptosis (TWEAK) is a mediator of inflammatory bone remodeling. The aim of this study was to investigate the role of TWEAK in modulating human osteoblast activity, and how TWEAK and TNFalpha might interact in this context. Recombinant TWEAK and TNF were both mitogenic for human primary osteoblasts (NHBC). TWEAK dose- and time-dependently regulated the expression of the osteoblast transcription factors RUNX2 and osterix. TWEAK inhibited in vitro mineralization and downregulated the expression of osteogenesis-associated genes. Significantly, TWEAK and TWEAK/TNF induced the expression of the osteoblast differentiation inhibitor and SOST gene product, sclerostin. Sclerostin induction was mitogen-activated protein kinase (MAPK) dependent. The SOST mRNA levels induced by TWEAK were equivalent to or exceeded those seen in steady-state human bone, and the TWEAK/TNF induction of SOST mRNA was recapitulated in fresh cancellous bone explants. TWEAK-induced sclerostin expression was observed in immature osteoblastic cells, both in cycling (Ki67(+)) primary NHBC and in the cell lines MC3T3-E1 and MG-63, as well as in human osteocyte-like cells and in the osteocyte cell line, MLO-Y4. Treatment of NHBC with recombinant human sclerostin mimicked the effects of TWEAK to suppress RUNX2 and osteocalcin (OCN). TWEAK, TNF, and sclerostin treatment of NHBC similarly altered levels of phosphorylated and total GSK3beta and active and total levels of beta-catenin, implying that the Wnt signaling pathway was affected by all three stimuli. Sclerostin also rapidly activated ERK-1/2 MAPK signaling, indicating the involvement of additional signaling pathways. Together, our findings suggest that TWEAK, alone and with TNF, can regulate osteoblast function, at least in part by inducing sclerostin expression. Our results also suggest new roles and modes of action for sclerostin.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Mediadores de Inflamación/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factores de Necrosis Tumoral/farmacología , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Western Blotting , Proteínas Morfogenéticas Óseas/genética , Proliferación Celular/efectos de los fármacos , Citocina TWEAK , Cartilla de ADN , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Marcadores Genéticos/genética , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Osteoblastos/citología , Osteoblastos/enzimología , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacosRESUMEN
Porous tantalum (Ta) has found application in orthopedics, although the interaction of human osteoblasts (HOB) with this material has not been reported. The aim of this study was to investigate the interaction of primary HOB with porous tantalum, using 5-mm thick discs of porous tantalum. Comparison was made with discs of solid tantalum and tissue culture plastic. Confocal microscopy was used to investigate the attachment and growth of cells on porous Ta, and showed that HOB attached successfully to the metal "trabeculae," underwent extensive cell division, and penetrated into the Ta pores. The maturation of HOB on porous Ta was determined in terms of cell expression of the osteoblast phenotypic markers, STRO-1, and alkaline phosphatase. Despite some donor-dependent variation in STRO-1/AlkPhos expression, growth of cells grown on porous Ta either promoted, or did not impede, the maturation of HOB. In addition, the expression of key osteoblastic genes was investigated after 14 days of culture. The relative levels of mRNA encoding osteocalcin, osteopontin and receptor activator of NFkappaB ligand (RANKL) was not different between porous or solid Ta or plastic, although these genes were expressed differently by cells of different donors. However, bone sialoprotein and type I collagen mRNA species showed a decreased expression on porous Ta compared with expression on plastic. No substrate-dependent differences were seen in the extent of in vitro mineralization by HOB. These results indicate that porous Ta is a good substrate for the attachment, growth, and differentiated function of HOB.
Asunto(s)
Materiales Biocompatibles , Osteoblastos/citología , Osteoblastos/fisiología , Fenotipo , Tantalio , Anciano , Anciano de 80 o más Años , Adhesión Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , PorosidadRESUMEN
Circulating 1 alpha,25-dihydroxyvitamin D(3) (1,25D) derives from renal conversion of 25-hydroxyvitamin D(3) (25D), by the 25D 1 alpha-hydroxylase (CYP27B1). Blood 25D levels, but not 1,25D levels, are the best indicator of vitamin D status and predict fracture risk in the elderly. We examined the extent to which osteoblasts can metabolize 25D. Well-characterized human primary osteoblasts and osteosarcoma (OS) cell lines were examined for the expression and regulation of genes associated with vitamin D metabolism, using real-time PCR. Primary osteoblasts and OS cell lines were found to express CYP27B1 mRNA and secreted detectable 1,25D in response to 25D. Of the OS cell lines tested, HOS expressed the most CYP27B1 mRNA and secreted the highest levels of 1,25D. All osteoblastic cells examined up-regulated expression of the catabolic regulator of 1,25D, the 25-hydroxyvitamin D-24-hydroxylase (CYP24), when incubated with either 1,25D or 25D. Exposure to physiological levels of 25D resulted in up-regulated transcription of the 1,25D responsive genes, osteocalcin (OCN), osteopontin (OPN) and RANKL. Specific knockdown of CYP27B1 in HOS cells using siRNA resulted in up to 80% reduction in both 1,25D secretion and the transcription of OCN and CYP24, strongly implying that the 25D effect in osteoblasts is preceded by conversion to 1,25D. Incubation with 25D, like 1,25D, inhibited primary osteoblast proliferation and promoted in vitro mineralization. Finally, we detected expression by osteoblasts of receptors for vitamin D binding protein (DBP), cubilin and megalin, suggesting that osteoblasts are able to internalize DBP-25D complexes in vivo. Together, our results suggest that autocrine, and perhaps paracrine, pathways of vitamin D(3) metabolism may regulate key osteoblast functions independently of circulating, kidney derived 1,25D. Our results are therefore consistent with the reported benefits of maintaining a healthy vitamin D status in the elderly to reduce the risk of fractures.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Calcifediol/metabolismo , Calcitriol/sangre , Colecalciferol/metabolismo , Osteoblastos/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Calcifediol/genética , Línea Celular Tumoral , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Osteocalcina/metabolismo , Osteopontina/metabolismo , Osteosarcoma/patología , Reacción en Cadena de la Polimerasa , Ligando RANK/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/metabolismo , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Transcripción Genética , Vitamina D3 24-HidroxilasaRESUMEN
Breast cancer is the most common carcinoma that metastasizes to bone. To examine the efficacy of recombinant soluble Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against breast cancer growth in bone, we established a mouse model in which MDA-MB-231 human breast cancer cells were transplanted directly into the marrow cavity of the tibiae of athymic nude mice producing osteolytic lesions in the area of injection. All vehicle-treated control animals developed large lesions that established in the marrow cavity, eroded the cortical bone, and invaded the surrounding soft tissue, as assessed by radiography, micro-computed tomography, and histology. In contrast, animals treated with recombinant soluble Apo2L/TRAIL showed significant conservation of the tibiae, with 85% reduction in osteolysis, 90% reduction in tumor burden, and no detectable soft tissue invasion. Tumor cells explanted from Apo2L/TRAIL-treated animals were significantly more resistant to the effects of Apo2L/TRAIL when compared with the cells explanted from the vehicle-treated control animals, suggesting that prolonged treatment with Apo2/TRAIL in vivo selects for a resistant phenotype. However, such resistance was readily reversed when Apo2L/TRAIL was used in combination with clinically relevant chemotherapeutic drugs, including taxol, etoposide, doxorubicin, cisplatin, or the histone deacetylase inhibitor suberoylanilide hydroxamic acid. These studies show for the first time that Apo2L/TRAIL can prevent breast cancer-induced bone destruction and highlight the potential of this ligand for the treatment of metastatic breast cancer in bone.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Glicoproteínas de Membrana/farmacología , Osteólisis/prevención & control , Factor de Necrosis Tumoral alfa/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/administración & dosificación , Neoplasias de la Mama/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Glicoproteínas de Membrana/administración & dosificación , Ratones , Ratones Desnudos , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
While the apoptosis-inducing ligand Apo2L/TRAIL is a promising new agent for the treatment of cancer, the sensitivity of cancer cells for induction of apoptosis by Apo2L/TRAIL varies considerably. Identification of agents that can be used in combination with Apo2L/TRAIL to enhance apoptosis in breast cancer cells would increase the potential utility of this agent as a breast cancer therapeutic. Here, we show that the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize Apo2L/TRAIL-resistant breast cancer cells to Apo2L/TRAIL-induced apoptosis. Importantly, neither Apo2L/TRAIL alone, nor in combination with SAHA, affected the viability of normal human cells in culture. Apo2L/TRAIL-resistant MDA-MB-231 breast cancer cells, generated by long-term culture in the continuous presence of Apo2L/TRAIL, were resensitized to Apo2L/TRAIL-induced apoptosis by SAHA. The sensitization of these cells by SAHA was accompanied by activation of caspase 8, caspase 9 and caspase 3 and was concomitant with Bid and PARP cleavage. The expression of the proapoptotic protein, Bax, increased significantly with SAHA treatment and high levels of Bax were maintained in the combined treatment with Apo2L/TRAIL. Treatment with SAHA increased cell surface expression of DR5 but not DR4. Interestingly, SAHA treatment also resulted in a significant increase in cell surface expression of DcR1. Taken together, our findings indicate that the use of these 2 agents in combination may be effective for the treatment of breast cancer.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Glicoproteínas de Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/metabolismo , Humanos , Receptores de Superficie Celular/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , VorinostatRESUMEN
While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1alpha,25-dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25-100 ng/ml) and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures, unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.
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Remodelación Ósea/fisiología , Huesos/metabolismo , Diferenciación Celular/fisiología , Osteoblastos/metabolismo , Osteoclastos/metabolismo , ADP-Ribosil Ciclasa/metabolismo , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Antígenos CD/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Huesos/citología , Huesos/efectos de los fármacos , Calcitriol/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/fisiología , Células Cultivadas , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero/farmacología , Dexametasona/farmacología , Proteínas Ligadas a GPI , Glicoproteínas/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Ratones , Osteoblastos/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoprotegerina , Hormona Paratiroidea/farmacología , Ligando RANK , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/genética , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células del Estroma/metabolismoRESUMEN
Tantalum (Ta) is increasingly used in orthopaedics, although there is a paucity of information on the interaction of human osteoblasts with this material. We investigated the ability of Ta to support the growth and function of normal human osteoblast-like cells (NHBC). Cell responses to polished and textured Ta discs were compared with responses to other common orthopaedic metals, titanium and cobalt-chromium alloy, and tissue culture plastic. No consistent differences, that could be attributed to the different metal substrates or to the surface texture, were found in several measured parameters. Attachment of NHBC to each substrate was similar, as was cell morphology, as determined by confocal microscopy. Cell proliferation was slightly faster on plastic than on Ta at 3 days, but by 7 days neither the absolute cell numbers, nor the number of cell divisions, was different between Ta and the other substrates. No consistent, substrate-dependent differences were seen in the expression of a number of mRNA species corresponding to the pro-osteoclastic or the osteogenic activity of osteoblasts. No substrate-dependent differences were seen in the extent of in vitro mineralisation by NHBC. These results indicate that Ta is a good substrate for the attachment, growth and differentiated function of human osteoblasts.
Asunto(s)
Materiales Biocompatibles/química , Calcio/metabolismo , Glicoproteínas/metabolismo , Sustancias de Crecimiento/metabolismo , Osteoblastos/citología , Osteoblastos/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Tantalio/química , Calcificación Fisiológica/fisiología , Adhesión Celular/fisiología , División Celular , Tamaño de la Célula , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Humanos , Ensayo de Materiales , Osteogénesis/fisiología , Osteoprotegerina , Fenotipo , Receptores del Factor de Necrosis Tumoral , Propiedades de SuperficieRESUMEN
SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY(246)AQL and VVY(263)ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34(+)CD38(+) and early clonogenic progenitors, and at lower levels on CD34(+)CD38(-) cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133(+) precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Delta 46-110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Células Madre Hematopoyéticas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Mesodermo/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Endotelio/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11RESUMEN
Five expert laboratories have participated in a cross-laboratory study to co-evaluate and compare three commercial Factor VIII/von Willebrand factor (VWF) concentrates. A total of nine factor concentrate lots were evaluated, comprising AHF (High Purity) (AHF HP; x3), Biostate (x3) and Humate/Haemate (x3). All laboratories blind tested for FVIII: C, VWF: Ag and VWF: CB, four tested for VWF: RCo, and one performed VWF: Multimers. The study yielded inter-laboratory CVs for VWF: Ag and FVIII:C around 10-15%, and for VWF:CB and VWF:RCo around 20%, significantly lower than those of previous multi-laboratory surveys. All three lots of AHF HP contained in the vicinity of 25 U/ml FVIII:C, around 60-75 U/ml of VWF:Ag, but only 30-45 U/ml of VWF:CB and 40-50 U/ml of VWF:RCo (thus, CB/Ag ratio around 0.5-0.6 and RCo/Ag ratio around 0.6-0.7). Study determined that FVIII: C and VWF: RCo levels were similar to manufacturer assigned levels. Some loss of the high molecular weight (HMW) multimers was observed, together with an intense low molecular weight (LMW) VWF band consistent with some reduction or proteolysis of HMW VWF. All three lots of Humate/Haemate contained in the vicinity of 23-32 U/ml of FVIII:C, 70-105 U/ml of VWF: Ag, 50-90 U/ml of VWF: CB and VWF: RCo (i.e. CB/Ag ratio around 0.6-0.9 and RCo/Ag ratio around 0.6-1.1). Study-determined FVIII: C and VWF: RCo levels were similar to manufacturer-assigned levels. The LMW multimer band seen with AHF HP was also observed with Humate/Haemate. All three lots of Biostate contained in the vicinity of 40-55 U/ml of FVIII:C, 105-170 U/ml of VWF:Ag, 90-150 U/ml of VWF:CB, and 90-135 U/ml of VWF:RCo (i.e. CB/Ag and RCo/Ag ratios around 0.7-1.0). Study-determined FVIII:C levels were similar to manufacturer-assigned levels. The LMW multimer band seen with AHF HP was not observed with Biostate. The defined pattern of increasing CB/ Ag from AHF HP to Humate/Haemate and Biostate was consistently observed in study data from each of the five laboratories. In conclusion, study findings indicate some differences in the retention of functional/ HMW VWF between factor concentrates, and this is expected to have significant implications in terms of clinical efficacy for therapy in VWD.