Cell-type-dependent up-regulation of in vitro mineralization after overexpression of the osteoblast-specific transcription factor Runx2/Cbfal.
ABSTRACT Functional expression of the transcriptional activator Runx2/Cbfal is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast-specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast-like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast-specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up-regulated expression in MC3T3-E1 immature osteoblast-like cells. However, Runx2 expression enhanced matrix mineralization in a cell-type-dependent manner. NIH3T3 and IMR-90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2-expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant-dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2-infected C3H10T1/2 cells compared with Runx2-expressing cultures which did not mineralize. Treatment of Runx2-infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast-like MC3T3-E1 cells resulted in acceleration and robust up-regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast-specific activities, in vitro matrix mineralization requires additional tissue-specific cofactors, which supplement Runx2 activity.
Article: Runx2 overexpression enhances osteoblastic differentiation and mineralization in adipose--derived stem cells in vitro and in vivo.[show abstract] [hide abstract]
ABSTRACT: Like bone marrow stromal cells, adipose tissue-derived stem cells (ADSCs) possess multilineage potential, a capacity for self-renewal and long-term viability. To confirm whether ADSCs represent a promising source of cells for gene-enhanced bone tissue-engineering, the osteogenic potential of ADSCs under the control of certain osteoinductive genes has been evaluated. Runx2, a transcription factor at the downstream end of bone morphogenetic protein (BMP) signaling pathways, is essential for osteoblast differentiation and bone formation. In this study we used adenovirus vector to deliver Runx2 to ADSCs and then examined the enhancement of osteogenic activity. Overexpression of Runx2 inhibited adipogenesis, as demonstrated by suppression of LPL and PPARgamma expression at the mRNA level and reduced lipid droplet formation. Moreover, ADSCs transduced with Ad-Runx2 underwent rapid and marked osteoblast differentiation as determined by osteoblastic gene expression, alkaline phosphatase activity and mineral deposition. Additionally, histological examination revealed that implantation of Runx2 modified ADSCs could induce mineral deposition and bone-like tissue formation in vivo. These results confirmed, firstly, the ability of Runx2 to promote osteogenesis and cell differentiation and, secondly, the competence of ADSCs as target cells for bone tissue engineering. Our work demonstrates a potential new approach for bone repair using Runx2-modified ADSCs for bone tissue engineering.Calcified Tissue International 10/2006; 79(3):169-78. · 2.38 Impact Factor
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ABSTRACT: The aim of this study was to study the effects of various surface treatments to a titanium surface on the expression of Runx2 in vitro. Human Osteosarcoma TE-85 cells were cultured on machined, sandblasted, or anodic oxidized cpTi discs. At various times of incubation, the cells were collected and then processed for the analysis of mRNA expression of Runx2 using reverse transcription-PCR. The expression pattern of Runx2 mRNA was differed according to the types of surface treatment. When the cells were cultured on the untreated control culture plates, the gene expression of Runx2 was not increased during the experiments. In the case of that the cells were cultured on the machined cpTI discs, the expression level was intermediate at the first day, but increased constitutively to day 5. In cells on sandblasted cpTi discs, the expression level was highest in the first day sample and the level was maintained to 5 days. In cells on anodized cpTi discs, the expression level increased rapidly to 3 days, but decreased slightly in the 5-th day sample. Different surface treatments may contribute to the regulation of osteoblast function by influencing the level of gene expression of key osteogenic factors.The journal of advanced prosthodontics 07/2009; 1(2):91-6.
Article: Autoregulatory mechanism of Runx2 through the expression of transcription factors and bone matrix proteins in multipotential mesenchymal cell line, ROB-C26.[show abstract] [hide abstract]
ABSTRACT: Runx2 is essential for osteoblast differentiation and gene expression of bone matrix proteins, however, little is known about the mechanism regulating its activity. In this study, the role of Runx2 on gene expression of transcription factors, AJ18, Msx2, and Dlx5, was examined in vitro. It is known that AJ18 and Msx2 act as repressors to inhibit activity of Runx2, whereas Dlx5 promotes its activity. An expression vector inserted Runx2 cDNA was transiently overexpressed in a rat multipotential mesenchymal cell line, ROB-C26 (C26). Real time reverse transcription-PCR analysis showed that, in exogenous Runx2-overexpressing C26 cells (C26-Rx), AJ18 expression increased 1.8-fold, Msx2 expression increased 3.0-fold, and Dlx5 expression increased 2.7-fold compared to the cells transfected with vector alone (C26-Co). Luciferase assay also showed that, in C26-Rx, AJ18 promoter activity increased 2.1-fold compared to C26-Co. Furthermore, gene expression of alkaline phosphatase (ALP) and bone matrix proteins including type I collagen (Col1), osteocalcin (OC), osteopontin (OPN), and matrix Gla protein (MGP) was examined. In C26-Rx, MGP expression increased 1.8-fold, and OPN expression increased 1.4-fold compared to C26-Co. However, no significant difference in Col1, ALP, and OC expressions was detected between C26-Rx and C26-Co. These results suggest that the existence of autoregulatory feed back loops, which inhibit Runx2 activity through the interaction of AJ18, Dlx5, and Msx2 cooperating with that of MGP and OPN, interferes with the differentiation of C26 cells toward mature osteoblasts.Journal of Oral Science 01/2006; 47(4):199-207.