ABSTRACT
La estimulación mecánica juega un papel fundamental en el mantenimiento de la masa ósea. Dicha estimulación previene la apoptosis de los osteocitos por un mecanismo que implica la acumulación de β-catenina y la translocación nuclear de quinasas reguladas por señales extracelulares (ERK). El factor de crecimiento del endotelio vascular (VEGF) y la proteína relacionada con la parathormona (PTHrP) modulan la formación ósea, aunque su interacción con los osteocitos es desconocida. En el presente estudio hemos evaluado el posible papel del receptor 2 del VEGF (VEGFR2) y del receptor tipo 1 de PTH (PTH1R) en la respuesta antiapoptótica a la estimulación mecánica en células osteocíticas MLO-Y4. Las células se sometieron a estrés mecánico por flujo laminar de fluido (10 min, 10 dinas/cm2) o choque hipotónico (240 mOsm, 1h), o estimuladas con VEGF165 o PTHrP (1-36). Además, comparamos los efectos de sobre-expresar VEGFR2 y el estímulo mecánico en estas células. La estimulación mecánica, el VEGF165 o la PTHrP (1-36), de manera similar, estimularon la viabilidad celular y la estabilización de β-catenina, relacionada con su localización en la membrana. Además, la estimulación mecánica aumentó la presencia del PTH1R en la membrana. La inhibición del VEGFR2 así como el antagonista PTHrP (7-34) disminuyeron estos efectos. Por otro lado, la sobre-expresión del VEGFR2 en las células MLO-Y4 mimetizó el efecto del estímulo mecánico sobre la β-catenina y la viabilidad celular. Estos hallazgos apoyan un papel funcional de ambos sistemas, VEGF/VEGFR2 y PTHrP/PTH1R, en la respuesta temprana a la estimulación mecánica para promover la viabilidad osteocítica (AU)
Mechanical stimulation plays a crucial role in bone mineral maintenance. This stimulation prevents osteocyte apoptosis by a mechanism that involves β-catenin accumulation and nuclear translocation of extracellular-signal-regulated kinases (ERKs). The vascular endothelial growth factor (VEGF) and parathyroid hormone-related protein (PTHrP) modulate bone formation, although their interaction with osteocytes is unknown. In this paper we have considered the possible role of VEGF (VEGFR2) 2 receptor and PTH (PTH1R) type 1 receptor in the anti-apoptotic response to mechanical stimulation of MLO-Y4 osteocyte-like cells. The cells were subjected to mechanical stress by laminar fluid flow (10 min, 10 dinas/cm2 ) or hypotonic shock (240 mOsm, 1h), or stimulated with VEGF165 or PTHrP (1-36). We also compared the effects of overexpressed VEGFR2 and mechanical stimulation of these cells. Mechanical stimulation, VEGF165 or PTHrP (1-36) stimulated cellular viability and β-catenin stabilization in a similar manner, associated with its localization in the membrane. Mechanical stimulation increased PTH1R presence in the membrane. VEGFR2 inhibition as well as the PTHrP (7-34) antagonist reduced these effects. On the other hand, VEGFR2 overexpression in MLO-Y4 cells mimicked the mechanical stimulation effect on β-catenin and cellular viability. Our findings support a functional role for both systems, VEGF/VEGFR2 and PTHrP/PTH1R, in the early response to mechanical stimulation in promoting osteocyte-like viability (AU)
Subject(s)
Humans , Osteoporosis/drug therapy , Vascular Endothelial Growth Factor Receptor-2/pharmacokinetics , Receptor, Parathyroid Hormone, Type 1/therapeutic use , Apoptosis , Osteoporosis/physiopathology , Osteocytes , beta Catenin/pharmacokineticsABSTRACT
El estrés oxidativo es clave en el envejecimiento y en los estados diabéticos. La carga mecánica es decisiva para mantener la masa ósea. La respuesta del hueso a los estímulos mecánicos parece reducirse con el envejecimiento y probablemente en la enfermedad ósea provocada por la diabetes. Entender los mecanismos mediante los que el estrés oxidativo afecta a la función de las células óseas, y en concreto a la mecanotransducción en el osteocito, podría proporcionar nuevas dianas moleculares para mejorar los tratamientos actuales y el diseño de otros nuevos para prevenir la pérdida de masa ósea. Nuestros resultados indican que un medio de alta glucosa («diabético») ejerce un efecto negativo sobre la capacidad de los osteocitos para responder a estímulos mecánicos, a través de la interacción con la β-catenina. Además, estos hallazgos sugieren que el estímulo mecánico promueve la viabilidad osteocítica, al menos en parte, a través de la producción de la proteína relacionada con la parathormona (PTHrP) (AU)
Oxidative stress is a key factor in aging and diabetes. Mechanical loading is critical to maintain bone mass. The response of bone to mechanical stimuli appears to be reduced with aging and probably in bone disease caused by diabetes. Understanding the mechanisms by which oxidative stress affects function of bone cells, and specifically to osteocyte mechanotransduction, may provide new molecular targets to improve current treatments and design new treatments to prevent bone loss. Our results indicate that high glucose medium («diabetic ») has a negative effect on the ability of osteocytes to respond to mechanical stimuli affecting b-catenin and apoptosis. Moreover, these findings suggest that the mechanical stimulus promotes viability osteocytic, at least in part, through production of parathyroid hormone related protein (PTHrP) (AU)
Subject(s)
Animals , Male , Female , Mice , Osteocytes , Osteocytes/radiation effects , Oxidative Stress/physiology , Diabetes Complications/physiopathology , Apoptosis , Cellular Apoptosis Susceptibility Protein/analysis , Blotting, Western/methods , Cell Death/physiology , 28599 , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/trendsABSTRACT
PTHrP is an important regulator of bone remodelling, apparently by acting through several sequence domains. We here aimed to further delineate the functional roles of the nuclear localization signal (NLS) comprising the 88-107 amino acid sequence of PTHrP in osteoblasts. PTHrP mutants from a human PTHrP (-36/+139) cDNA (wild type) cloned into pcDNA3.1 plasmid with deletion (Δ) of the signal peptide (SP), NLS, T(107), or T107A replacing T(107) by A(107) were generated and stably transfected into osteoblastic MC3T3-E1 cells. In these cells, intracellular trafficking, cell proliferation and viability, as well as cell differentiation were evaluated. In these transfected cells, PTHrP was detected in the cytoplasm and also in the nucleus, except in the NLS mutant. Meanwhile, the PTH type 1 receptor (PTH1R) accumulates in the cytoplasm except for the ΔSP mutant in which the receptor remains at the cell membrane. PTHrP-wild type cells showed enhanced growth and viability, as well as an increased matrix mineralization, alkaline phosphatase activity, and osteocalcin gene expression; and these features were inhibited or abolished in ΔNLS or ΔT(107) mutants. Of note, these effects of PTHrP overexpression on cell growth and function were similarly decreased in the ΔSP mutant after PTH1R small interfering RNA transfection or by a PTH1R antagonist. The present in vitro findings suggest a mixed model for PTHrP actions on osteoblastic growth and function whereby this protein needs to be secreted and internalized via the PTH1R (autocrine/paracrine pathway) before NLS-dependent shuttling to the nucleus (intracrine pathway).