RESUMEN
Cells, particularly mechano-sensitive musculoskeletal cells such as tenocytes, routinely encounter oxidative stress. Oxidative stress can not only stimulate tissue repair, but also cause damage leading to tissue degeneration. As diabetes is associated with increased oxidative damage as well as increased risk of tendon degeneration, the aim of this study was to determine if extracellular glucose levels alter the response of tendon cells to oxidative stress. Primary human tenocytes were cultured in either high (17.5 mM) or low (5 mM) glucose and treated with 100 µM hydrogen peroxide. In low glucose, peroxide-treated cells remained fully viable and collagen synthesis was increased, suggesting an anabolic response. In high glucose, however, peroxide treatment led to increased bim-mediated apoptosis. The activities of both forkhead box O (FOXO1) and p53 were required for upregulation of bim RNA expression in high glucose. We found that both p53-mediated inhibition of the bim repressor micro RNA (miR17-92) and FOXO1-mediated upregulation of bim transcription were required to permit accumulation of bim RNA. High glucose coupled with oxidative stress resulted in upregulation of miR28-5p, which directly inhibited expression of the p53 deacetylase sirtuin 3, resulting in increased levels of acetylated p53. In peroxide-treated cells in both high and low glucose, protein levels of acetylated FOXO1 as well as HIF1α (hypoxia-inducible factor 1α) were increased. However, under low-glucose conditions, peroxide treatment resulted in activation of p38, which inhibited FOXO1-mediated but promoted HIF1α-mediated transcriptional activity. In low glucose, HIF1α upregulated expression of sox9 and scleraxis, two critical transcription factors involved in establishing the tenocyte phenotype, and increased collagen synthesis. The switch from FOXO1-mediated (proapoptosis) to HIF1α-mediated (prodifferentiation) transcription occurred at an extracellular glucose concentration of 7 mM, a concentration equivalent to the maximum normal blood glucose concentration. Extracellular glucose has a profound effect on the cellular response to oxidative stress. A level of oxidative stress normally anabolic may be pathological in high glucose.
Asunto(s)
Apoptosis , Diferenciación Celular , Glucosa/metabolismo , Estrés Oxidativo , Tendones/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína 11 Similar a Bcl2 , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Activación Enzimática , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Glucosa/deficiencia , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Largo no Codificante , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Sirtuina 3/genética , Sirtuina 3/metabolismo , Tendones/efectos de los fármacos , Tendones/patología , Transcripción Genética , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: To identify osteoarthritis (OA) relevant genes and pathways in damaged and undamaged cartilage isolated from the knees of patients with anteromedial gonarthrosis (AMG) - a specific form of knee OA. DESIGN: Cartilage was obtained from nine patients undergoing unicompartmental knee replacement (UKR) for AMG. AMG provides a spatial representation of OA progression; showing a reproducible and histologically validated pattern of cartilage destruction such that damaged and undamaged cartilage from within the same knee can be consistently isolated and examined. Gene expression was analysed by microarray and validated using real-time PCR. RESULTS: Damaged and undamaged cartilage showed distinct gene expression profiles. 754 genes showed significant up- or down-regulation (non-False discovery rate (FDR) P < 0.05) with enrichment for genes involved in cell signalling, Extracellular Matrix (ECM) and inflammatory response. A number of genes previously unreported in OA showed strongly altered expression including RARRES3, ADAMTSL2 and DUSP10. Confirmation of genes previously identified as modulated in OA was also obtained e.g., SFRP3, MMP3 and IGF1. CONCLUSIONS: This is the first study to examine a common and consistent phenotype of OA to allow direct comparison of damaged and undamaged cartilage from within the same joint compartment. We have identified specific gene expression profiles in damaged and undamaged cartilage and have determined relevant genes and pathways in OA progression. Importantly this work also highlights the necessity for phenotypic and microanatomical characterization of cartilage in future studies of OA pathogenesis and therapeutic development.
Asunto(s)
Cartílago Articular/metabolismo , Osteoartritis de la Rodilla/genética , Transcriptoma/fisiología , Anciano , Artroplastia de Reemplazo de Rodilla , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/cirugía , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff. We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1alpha (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1alpha regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis). The HIF-1alpha expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears. These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.
Asunto(s)
Lesiones del Manguito de los Rotadores , Síndrome de Abducción Dolorosa del Hombro/patología , Tendinopatía/patología , Adolescente , Adulto , Anciano , Apoptosis , Hipoxia de la Célula , Fragmentación del ADN , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/metabolismo , Manguito de los Rotadores/metabolismo , Manguito de los Rotadores/patología , Síndrome de Abducción Dolorosa del Hombro/metabolismo , Tendinopatía/metabolismo , Adulto JovenRESUMEN
Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times. This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured.
Asunto(s)
Cartílago Articular , Condrocitos , Criopreservación , Anciano , Anciano de 80 o más Años , Cartílago Articular/citología , Cartílago Articular/metabolismo , Proliferación Celular , Supervivencia Celular , Condrocitos/citología , Condrocitos/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Osteoblasts undergo apoptosis both in vitro and in vivo in response to high dose glucocorticoid (GC) treatment. However, the molecular mechanisms remain elusive, hindering the prevention and treatment of this side-effect. Apoptosis was induced by dexamethasone (Dex) in murine MBA-15.4 osteoblasts within 24-48 h of treatment. We found dose- and time-dependent upregulation of Bim protein, a pro-apoptotic Bcl-2 family member, with highest levels at 24-48 h for 1 microM Dex. This was also observed in primary human bone marrow stromal cells. Bim is subjected to stringent transcriptional and post-translational regulation in osteoblasts. Bim mRNA was upregulated in response to 1 microM Dex; both cycloheximide and the GC receptor antagonist, RU486, prevented Dex-induction of Bim protein, indicating transcriptional regulation involving the GC receptor. The proteasome inhibitor, MG132, potently increased Bim protein levels. Bim was also upregulated in osteoblasts undergoing apoptosis in response to serum deprivation and matrix detachment. Gene silencing experiments show that short interference RNA (siRNA) specific for Bim or the downstream effector Bax both reduced apoptosis induced by Dex in osteoblastic cells. These findings suggest that Bim is a novel regulator of osteoblast apoptosis and may be a therapeutic target.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Células de la Médula Ósea/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Proteínas de la Membrana/metabolismo , Osteoblastos/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Células del Estroma/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Dexametasona/administración & dosificación , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Glucocorticoides/administración & dosificación , Humanos , Leupeptinas/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Mifepristona/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Receptores de Glucocorticoides/antagonistas & inhibidores , Células del Estroma/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba , Proteína X Asociada a bcl-2/genéticaRESUMEN
This article reports the mechanical properties and in vitro evaluation of a collagen scaffold fabricated using an indirect 3D printing technique. Collagen scaffolds, featuring predefined internal channels and capillary networks, were manufactured using phase change printing. It was observed that the collagen scaffolds featured internal channels and a hierarchical structure that varied over length scales of 10-400 microm. In vitro evaluation using hMSCs demonstrated that the resultant collagen based scaffolds have the ability to support hMSC cell attachment and proliferation; cells can migrate and survive deep within the structure of the scaffold. The cell numbers increased 2.4 times over 28 days in culture for the lysine treated scaffolds. The cells were spread along the collagen fibers to form a 3D structure and extracellular matrix was detected on the surface of the scaffolds after 4 weeks in culture. The crosslinking treatment enhanced the biostability and dynamic properties of the collagen scaffolds significantly.
Asunto(s)
Colágeno , Ingeniería de Tejidos , Animales , Bovinos , Proliferación Celular , Células Cultivadas , Humanos , Ensayo de Materiales/métodos , Ingeniería de Tejidos/métodosRESUMEN
Skeletal mass is maintained by a balance between formation and resorption, cell proliferation and apoptosis. In vitro, glucocorticoids (GCs) decrease extracellular signal-regulated kinases (ERK) activation by mitogens, thus inhibiting osteoblast proliferation. Both ERK activity and proliferation are restored by co-treatment with the protein tyrosine phosphatase inhibitor, vanadate. Since ERK signalling may also be anti-apoptotic, we explored the effects of vanadate on GC-induced apoptosis in vitro and in vivo. Apoptosis in MBA-15.4 pre-osteoblasts increased from 6 h and remained up to eightfold higher through 6 days of 10(- 6) M dexamethasone (Dex) treatment. Co-incubation with 10(- 7) M vanadate markedly reduced apoptosis at all time points. Vanadate also prevented GC-induced poly-ADP-ribose polymerase cleavage. We assessed the transcriptional profiles of seven anti-apoptotic proteins (Bcl-2, Bcl-X(L), inhibitors of apoptosis protein-1 (IAP-1), IAP-2, X-linked IAP (XIAP), Fas-associated death-domain-like IL-1beta-converting enzyme-inhibitory protein (FLIP(Long)) and FLIP(Short)) in osteoblasts subjected to various stimuli using real-time quantitative PCR. Although these anti-apoptotic genes responded to different mitogenic conditions, Dex failed to repress their expression, and in fact significantly up-regulated Bcl-X(L), IAP-2 and XIAP. Dex may therefore induce apoptosis by up-regulating pro-apoptotic gene expression. We have previously demonstrated that rats treated with GC develop low formation osteoporosis (bone histomorphometry and DEXA) and skeletal fragility (breaking strength) that were largely prevented by co-treatment with vanadate. We report here that vertebrae from rats treated with 3.5 mg/kg per day methylprednisolone for 9 weeks showed increased incidence of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling-positive apoptotic osteocytes, which was reduced by vanadate co-treatment. We conclude that vanadate prevents GC-induced apoptosis of pre-osteoblasts in vitro and osteocytes in vivo, and this may contribute to its bone-sparing effects in vivo.
Asunto(s)
Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Glucocorticoides/farmacología , Osteoblastos/fisiología , Osteocitos/fisiología , Vanadatos/farmacología , Animales , Apoptosis/fisiología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/genética , Medio de Cultivo Libre de Suero/farmacología , Expresión Génica/efectos de los fármacos , Glucocorticoides/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metilprednisolona/farmacología , Ratones , Mitógenos/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas/genética , Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Columna Vertebral/fisiología , Células Madre/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Transcripción GenéticaRESUMEN
BACKGROUND: Khellin is a naturally occurring furochromone which, when combined with artificial ultraviolet (UV) A or solar irradiation (KUVA), is reported to repigment vitiligo skin as effectively as PUVA photochemotherapy. The exact mechanism of KUVA-induced repigmentation is unknown. OBJECTIVES: The aim of this study was to test the effect of khellin and KUVA on proliferation and melanogenesis of normal human melanocytes and Mel-1 melanoma cells in vitro. METHODS: Cultured normal human melanocytes, Mel-1 melanoma cells and fibroblasts were treated with khellin, UVA and KUVA and the effect on proliferation determined by cell counting. The effect on melanogenesis was determined by a radiometric melanin formation assay. Changes in gene expression and protein synthesis were determined by Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses. RESULTS: Khellin stimulated proliferation of Mel-1 melanoma cells and melanocytes at concentrations between 1 nmol L-1 and 0.5 mmol L-1 with a peak effect at 0.01 mmol L-1 khellin. In contrast, khellin inhibited proliferation of fibroblasts over the entire concentration range tested. At concentrations above 0.5 mmol L-1, khellin was cytotoxic to both melanocytic cells and fibroblasts. Exposure of khellin-treated cells to single doses of UVA between 150 and 280 mJ cm-2 resulted in an enhanced proliferative effect. Khellin and KUVA also stimulated the melanogenic enzyme activity of pigmented cells, with the most effective treatment being 0.01 mmol L-1 khellin with 250 mJ cm-2 UVA. Western blot, Northern blot and RT-PCR analysis revealed that these increases in melanogenic enzyme activity were not due to increases in gene expression or protein synthesis. UVA treatment resulted in an increase in enzyme glycosylation and this correlated with the increase in melanogenesis. CONCLUSIONS: We conclude that khellin activated by UVA stimulates melanocyte proliferation and melanogenesis. Our results point to the possibility that current treatment regimens might be improved if reduced khellin doses are applied and suggest that improved delivery vehicles be tested.
Asunto(s)
Khellin/farmacología , Melanocitos/efectos de los fármacos , Melanoma/patología , Neoplasias Cutáneas/patología , Rayos Ultravioleta , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Humanos , Melaninas/biosíntesis , Melanocitos/citología , Melanocitos/efectos de la radiación , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismoRESUMEN
In vitro studies have shown that the phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA) induces neural crest cell differentiation into melanocytes, and stimulates proliferation and differentiation of normal melanocytes. As TPA is not a physiological agent, its action is clearly mimicking some in vivo pathway involved in these processes. An understanding of the effect of TPA on the expression of melanogenic genes will therefore provide valuable insight into the molecular mechanisms regulating melanocyte differentiation. In this study, we utilized primary cultures of neural crest cells and an immortalized melanocyte cell line (DMEL-2) which proliferates in the absence of TPA, to explore the effects of TPA on key melanogenic effectors. In neural crest cells, TPA was found to be necessary for both microphthalmia associated transcription factor (Mitf) up-regulation and for melanin synthesis. Using northern blots, we show that in DMEL-2 cells, TPA significantly increases the messenger ribonucleic acid (mRNA) levels of the tyrosinase gene family (tyrosinase, Tyrp1 and Dct) and the expression of Mitf. Western blots demonstrate that in these TPA-treated cells there is a concomitant increase in Tyr, Tyrp1 and glycosylated Dct protein levels. Pax3, a known Mitf regulator, is unaltered by TPA treatment. This study demonstrates the utility of a novel cell line for investigating the long-term effects of TPA on melanogenesis and provides an understanding of how TPA enhances mouse melanocyte differentiation.
Asunto(s)
Melanocitos/efectos de los fármacos , Melanocitos/fisiología , Cresta Neural/efectos de los fármacos , Cresta Neural/fisiología , Oxidorreductasas , Acetato de Tetradecanoilforbol/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Melanocitos/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Cresta Neural/citología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A central feature of glucocorticoid (GC)-induced osteoporosis is decreased bone formation, secondary to decreased numbers of functional osteoblasts. We find that ERK activity is essential for serum-induced osteoblast proliferation in vitro because inhibition of MAPK/ERK kinase activity by U0126 completely abolished both serum-induced activation of ERK and proliferation of mouse (MBA-15.4) and human (MG-63) osteoblast cell lines. Dexamethasone (Dex) rapidly (<2 h) inhibits the sustained phase of ERK activation, required for nuclear shift and mitogenesis. This inhibition is reversed by cotreatment with the protein synthesis inhibitor, cycloheximide, and by the GC receptor antagonist, RU486, suggesting a classical transcriptional mechanism. Phosphatase activity was up-regulated by Dex treatment, and inhibition of ERK activity by Dex was also reversed by the protein tyrosine phosphatase inhibitor, vanadate. Coupled with the rapidity of Dex action, this indicates immediate-early gene phosphatase involvement, and we therefore used quantitative, real-time PCR to examine expression profiles of the dual-specificity MAPK phosphatases, MKP-1 and MKP-3. MKP-1, but not MKP-3, mRNA expression was 10-fold up-regulated in both mouse and human osteoblast cell lines within 30 min of Dex treatment and remained elevated for 24 h. MKP-1 protein was also markedly up-regulated following 1-8 h of Dex treatment, and this correlated precisely with dephosphorylation of ERK. Cell proliferation was impaired by Dex treatment, and this was reversed by both RU486 and vanadate. Therefore, MKP-1 up-regulation provides a novel and rapid mechanism, whereby GCs inhibit osteoblast proliferation.
Asunto(s)
Proteínas de Ciclo Celular , Dexametasona/farmacología , Glucocorticoides/farmacología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoblastos/enzimología , Fosfoproteínas Fosfatasas , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Proteínas Sanguíneas/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Citosol/enzimología , Regulación hacia Abajo/fisiología , Fosfatasa 1 de Especificidad Dual , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/fisiología , Antagonistas de Hormonas/farmacología , Humanos , Ratones , Mifepristona/farmacología , Osteoblastos/citología , Fosforilación , Proteína Fosfatasa 1 , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Glucocorticoid-induced osteoporosis is characterized by decreased osteoblast numbers and a marked impairment of new bone formation. We found that, in vitro, dexamethasone inhibits both preosteoblast proliferation and mitogenic kinase activity in response to mitogens, and that inhibition of protein tyrosine phosphatases (PTPs) using sodium orthovanadate prevents this. Therefore, dexamethasone may act by either upregulating antiproliferative PTPs or downregulating promitogenic tyrosine-phosphorylated substrates. In this study, osteoporosis was induced in 3.5-month-old rats by subcutaneous injection with methylprednisolone 3.5 mg/kg per day for 9 weeks. Rats were treated with steroid alone or in combination with 0.5 mg/mL sodium orthovanadate, administered continuously in drinking water. Steroid-treated bones were significantly (p < 0.005) osteopenic (according to dual-energy X-ray absorptiometry) and physically weaker (p < 0.05) than controls. Quantitative bone histology confirmed a significant decrease in osteoid surfaces (p < 0.001), osteoblast numbers (p < 0.05), and rate of bone formation (p < 0.001). Concomitant treatment with vanadate largely prevented the densitometric, histologic, and physical abnormalities induced by prednisolone. This study supports our finding that PTPs are central to the negative regulation of osteoblast proliferation by glucocorticoids and, furthermore, suggests that PTP inhibitors such as sodium orthovanadate should be considered as novel anabolic agents for the treatment of steroid-induced osteoporosis.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Glucocorticoides/toxicidad , Osteoporosis/enzimología , Osteoporosis/prevención & control , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Vanadatos/uso terapéutico , Animales , Densidad Ósea/efectos de los fármacos , Densidad Ósea/fisiología , Inhibidores Enzimáticos/farmacología , Femenino , Metilprednisolona/toxicidad , Osteoporosis/inducido químicamente , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Ratas Sprague-Dawley , Vanadatos/farmacologíaRESUMEN
Statins have been postulated to affect bone metabolism. We investigated the effects of different doses of simvastatin (1, 5, 10, and 20 mg. kg(-1). d(-1)), atorvastatin (2.5 mg. kg(-1). d(-1)), and pravastatin (10 mg. kg(-1). d(-1)) administered orally for 12 weeks to intact female Sprague-Dawley rats and the effect of 20 mg. kg(-1). d(-1) simvastatin in sham-operated and ovariectomized rats on femoral bone mineral density (BMD) and quantitative bone histomorphometry (QBH) and compared them with controls. BMD was decreased by 1 mg. kg(-1). d(-1) simvastatin (P=0.042), atorvastatin (P=0.0002), and pravastatin (P=0.002). The effect on QBH parameters differed with different doses of simvastatin (ANOVA, P=0.00012). QBH parameters of both bone formation and resorption were equivalently and markedly increased by 20 mg. kg(-1). d(-1) simvastatin in 2 separate groups of intact rats and were reflected by a relatively unchanged BMD. At lower doses, 1 mg. kg(-1). d(-1) simvastatin decreased bone formation while increasing bone resorption, as reflected by a marked decrease in BMD. Ovariectomized animals receiving 20 mg. kg(-1). d(-1) simvastatin showed no change in BMD relative to the untreated, ovariectomized controls; their increase in bone formation was smaller than in sham-operated rats receiving simvastatin, and there was no change in bone resorption. Dose-response curves of simvastatin for bone formation and resorption differed. These studies indicate that (1) statins decrease BMD in rodents, (2) high-dose simvastatin increases bone formation and resorption, (3) low-dose simvastatin decreases bone formation and increases bone resorption, (4) the effects of simvastatin on QBH differ at different dosages, (5) the effects of simvastatin seen in intact rats are not observed in ovariectomized rats, and (6) simvastatin is unable to prevent bone loss caused by ovariectomy.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pravastatina/farmacología , Pirroles/farmacología , Simvastatina/farmacología , Animales , Atorvastatina , Resorción Ósea/diagnóstico por imagen , Resorción Ósea/etiología , Relación Dosis-Respuesta a Droga , Femenino , Ácidos Heptanoicos/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Ovariectomía , Pravastatina/administración & dosificación , Pirroles/administración & dosificación , Radiografía , Ratas , Ratas Sprague-Dawley , Simvastatina/administración & dosificaciónRESUMEN
Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We found that although the glucocorticoid, dexamethasone, stimulated osteoblast maturation, it also inhibited proliferation of a preosteoblastic cell line, MBA-15.4. The dexamethasone-induced decline in preosteoblast proliferation correlated with a 30-40% reduction in protein kinase C/TPA-stimulated mitogen-activated protein kinase (MAPK) activity. These steroid effects only became evident after 6-24 h treatment, implying that dexamethasone acts on de novo synthesis of proteins. Because MAPK is inactivated by dephosphorylation of tyrosine and threonine residues, cells were treated concomitantly for 24 h with dexamethasone and inhibitors of tyrosine (sodium orthovanadate) and/or serine/threonine phosphatases (sodium fluoride). MAPK activity and cell proliferation were restored when MBA-15.4 cells were treated with vanadate, suggesting that dexamethasone up-regulates tyrosine phosphatase activity. Inactivation of serine/threonine phosphatases with sodium fluoride had no effect. Inhibition of the PKA pathway (which is growth inhibitory in mature osteoblasts) with H-89 did not reverse the effects of dexamethasone. Pretreatment with dexamethasone inhibited both peak- and extended activation plateau-phases of MAPK activity. Both phases were fully restored by pretreatment with vanadate, implicating more than one tyrosine phosphatase. Cycloheximide, alone or in combination with dexamethasone, prevented drop-off from plateau to basal levels, suggesting that an inducible dual-specificity phosphatase regulates the plateau-phase. We conclude that dexamethasone may inhibit preosteoblast growth via a novel tyrosine phosphatase pathway.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , División Celular/efectos de los fármacos , Dexametasona/farmacología , Inhibidores Enzimáticos/farmacología , Osteoblastos/citología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Línea Celular , Cicloheximida/farmacología , Glucocorticoides/farmacología , Cinética , Ratones , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Células Madre/citología , Acetato de Tetradecanoilforbol/farmacología , Vanadatos/farmacologíaRESUMEN
The microenvironment is thought to play a key role in the control of neural crest cell diversification. To investigate its role in melanocyte differentiation we mapped the temporal and spatial distribution of pigmented melanocytes in embryonic chick skin and determined, by experimental means, the route taken by migrating melanocytes in the skin. We show that the New Hampshire Red/Black Australorp crossbreed exhibits melanization from 5 days of incubation (2 1/2 days earlier than is reported in other breeds). Contrary to previous reports our findings show that melanization is at first predominantly dermal. Both dermal and epidermal melanocyte numbers increase until Day 8, whereafter there is a dramatic decline in dermal melanocytes and by Day 10, melanocytes are almost exclusively located in the epidermis. Using homeotypic and heterotypic combinations of white and red/black dermis and epidermis we have demonstrated that premelanocytes arrive in the dermis of the trunk by Day 3 and begin to move into the epidermis from Day 4 onward. Results from these grafts and from tritium labeling studies strongly suggest that there is little or no reverse migration of premelanocytes from epidermis to dermis. Our findings indicate that overt melanocyte differentiation is not dependent on location in an epidermal environment, and that melanogenesis does not signify the end-stage in the migration process. Further, they suggest that the early dermal mesenchyme plays a key role in controlling melanogenesis.