RESUMEN
This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Hesperidina/farmacología , Osteogénesis/efectos de los fármacos , Animales , Proteína Morfogenética Ósea 2/farmacología , Regeneración Ósea , Línea Celular , Células Cultivadas , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , RatasRESUMEN
We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb(+)). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts and that survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3ß pathway supports this observation. Furthermore, this was abrogated by the addition of recombinant OA to cultures, which restored osteoclastogenesis to wild-type levels. Moreover, mix and match co-cultures demonstrated an induction of osteoclastogenesis in D2J osteoblasts co-cultured with osteoclasts of D2J or wild-type. Last, in functional osteo-assays, we show that bone resorption activity of D2J osteoclasts is dramatically reduced, and these osteoclasts present an abnormal ruffled border over the bone surface. Collectively, these data support a model whereby OA/Gpnmb acts as a negative regulator of osteoclast differentiation and survival but not function by inhibiting the ERK/AKT signaling pathways.
Asunto(s)
Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Mutación , Osteoclastos/citología , Ligando RANK/fisiología , Animales , Remodelación Ósea , Ratones , Ratones Endogámicos DBA , Ligando RANK/metabolismo , Transducción de Señal , Microtomografía por Rayos XRESUMEN
We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-ß receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-ß signaling. These data confirm the anabolic role of OA in postnatal bone formation.
Asunto(s)
Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Osteoblastos/fisiología , Osteocalcina/genética , Osteogénesis/genética , Transducción de Señal , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Huesos/metabolismo , Huesos/patología , Diferenciación Celular/genética , Masculino , Ratones , Ratones Endogámicos DBA , Mutación , Osteoblastos/citología , Fenotipo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests that osteoactivin is crucial for the differentiation and functioning of different cell types, including bone-forming osteoblasts and bone-resorbing osteoclast cells. Here, we review the literature to date on various regulatory functions of osteoactivin, as well as its discovery, structure, expression, and function in different tissues and cells. The transcriptional regulation of osteoactivin and its mechanism of action in normal and diseased conditions with special emphasis on bone are also covered in this review. In addition, we touch on the therapeutic potential of osteoactivin in cancer and bone diseases.
Asunto(s)
Huesos/fisiología , Proteínas del Ojo/fisiología , Glicoproteínas de Membrana/fisiología , Animales , Proteínas del Ojo/química , Proteínas del Ojo/genética , Humanos , Inflamación/fisiopatología , Hígado/fisiología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Músculo Esquelético/fisiología , Neoplasias/fisiopatología , Procesamiento Proteico-Postraduccional , Homología Estructural de ProteínaRESUMEN
Increasing osteoblast activity in an anabolic fashion may offer an ideal therapeutic treatment for various orthopedic complications including osteoporosis. The purpose of this study was to evaluate the effect of mevinolin, a clinical statin drug, on osteoblast function (MG 63 Cell Line) and compare its mode of action with the conventionally utilized parathyroid hormone (PTH). MG63 cells were treated with different concentrations (control, low (100 nM), medium (1 uM), and high (10 uM)) of mevinolin or Parathyroid hormone. The cells were incubated for 24, 48, and 72 hours at 37 degrees C in a 95% air and 5% CO2 environmental chamber. Data obtained in this study revealed that: (I) there were significant decreases in cell number after 24 hours upon the exposure of medium and high doses of mevinolin, (II) cell numbers rebounded back toward control after 48 hours and were similar in number at 72 hours, and (III) there were no significant changes in calcium or alkaline phosphatase activity were observed throughout the study. Morphologically, the cells treated with various doses of Mevinolin expressed similar structural changes to those observed using PTH. These changes included pleomorphic characteristics and an occasional hyperchromatic pattern during the entire duration of the study (72 Hours). Other structural features observed were spindle shapes, cluster arrangements and multinucleation. The majority of cells had multiple nucleoli in all treated groups compared to controls. The overall conclusion of this investigation demonstrated that the concentrations used (100 nM and 10 microM) did not appear to affect the mitotic activities of immature phenotypic MG-63 cells. In addition, the concentrations of mevinolin used did not trigger the differentiation process of the cells throughout the experimental phases. This observation led us to suggest that the reason for such an outcome could be attributed to the lack of a response in calcium production or alkaline phosphatase activity (stimulator to differentiation and mineralization process).