RESUMEN
CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.
Asunto(s)
Regeneración Ósea , Proteína Hiperexpresada del Nefroblastoma/biosíntesis , Osteoblastos/metabolismo , Regulación hacia Arriba , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Animales , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Ratones , Ratones Noqueados , Proteína Hiperexpresada del Nefroblastoma/genética , Osteoblastos/patología , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Proteína Smad1/biosíntesis , Proteína Smad1/genética , Proteína Smad5/biosíntesis , Proteína Smad5/genética , Factor de Transcripción Sp7 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Microtomografía por Rayos XRESUMEN
We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.
Asunto(s)
Acer/química , Proteínas Morfogenéticas Óseas/biosíntesis , Regeneración Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diarilheptanoides/farmacología , Osteoblastos/efectos de los fármacos , Éteres Fenílicos/farmacología , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Diarilheptanoides/química , Diarilheptanoides/aislamiento & purificación , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Éteres Fenílicos/química , Éteres Fenílicos/aislamiento & purificación , ARN Mensajero/biosíntesis , Factor de Transcripción Sp7 , Factores de Transcripción/biosíntesisRESUMEN
CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells.