RESUMEN
The low density lipoprotein receptor-related protein 5 (LRP5) is a key determinant of bone mass, via the Wnt signaling pathway control of osteoblast function. This study examined human LRP5 signaling and the effects of an intracellular domain single nucleotide polymorphism (SNP: p.V1525A) on osteoblast differentiation and mineralization. Constitutively active LRP5 was constructed by deletion of the extracellular domain of LRP5 (LRP5DeltaN). Expression of LRP5DeltaN-V, which carries the allele p.1525V, induced higher beta-catenin/TCF-LEF activity compared to LRP5DeltaN-A, which carries the allele p.1525A. In a yeast two-hybrid assay, LRP5DeltaN-V also demonstrated a stronger interaction with AXIN than LRP5DeltaN-A. Expression of either of the alleles did not change cell proliferation. However, cells expressing LRP5DeltaN-V showed increased alkaline phosphatase activity and bone nodule formation compared to cells transfected with empty vector or LRP5DeltaN-A after osteogenic supplement (OS: beta-glycerophosphate and l-ascorbic acid) treatment. Cells expressing LRP5DeltaN-V revealed significantly increased bone sialoprotein (BSP) expression after 7 days of OS treatment and maintained elevated expression until day 21. Osteocalcin (OCN) mRNA levels were increased after 14-21 days of OS treatment in LRP5DeltaN-V expressing cells. LRP5DeltaN-V expressing cells demonstrated positive interaction with BMP-2 signaling of transcription at the SBE-luc promoter. LRP5 signaling is affected by the cytoplasmic SNP, p.V1525A. mRNA levels of Runx2 and Osterix were not affected by this SNP.
Asunto(s)
Diferenciación Celular/genética , Proteínas Relacionadas con Receptor de LDL/genética , Osteoblastos/citología , Polimorfismo de Nucleótido Simple , Proteínas Wnt/metabolismo , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/metabolismo , Alelos , Proteína Axina , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Citoplasma/metabolismo , Humanos , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Eliminación de Secuencia , Transducción de Señal , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Técnicas del Sistema de Dos Híbridos , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow-derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Ingeniería de Tejidos/métodos , Vitamina D/farmacología , Vitaminas/farmacología , Adulto , Animales , Fosfatos de Calcio , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Durapatita , Humanos , Implantes Experimentales , Ratones , Ratones SCID , Osteoblastos/citologíaRESUMEN
Fetal bovine mandible-derived osteoblasts were cultured for the purpose of obtaining a spatiotemporal assessment of bone matrix protein expression during in vitro differentiation. The results obtained from electron microscopic, immunohistological, biochemical, and molecular biological analyses indicated that these primary cultured osteoblasts produce an abundant extracellular matrix which mineralizes during a 14-day culture period. During this process, a restricted, spatiotemporal pattern of bone sialoprotein expression was indicated by immunohistological and molecular evaluations. To test the possibility that bone sialoprotein promoted the continued morphodifferentiation of osteoblastic cells, cultures were grown in the presence of anti-bone sialoprotein antibodies known to interfere with cell-bone sialoprotein attachment. Compared with cultures grown in the presence of normal rabbit serum (1:150), cultures grown in the media containing anti-bone sialoprotein antibody (1:150) failed to mineralize as demonstrated by von Kossa staining and failed to express osteocalcin and osteopontin as shown by the reverse transcription polymerase chain reaction. These results contribute to the growing evidence that bone sialoprotein is an important determinant of osteoblast differentiation and bone formation. Matrix protein-cell interactions may be examined using this spatiotemporally defined model.