Abstract
BackgroundNumerous transcription factors are involved in the establishment and maintenance of the osteoblastic phenotype, such as Runx2, osterix and Dlx5. The transcription factor retinoblastoma binding protein-1 (RBP1) was recently identified as an estrogen regulated gene in an osteosarcoma cell model. Since the function of RBP1 in osteoblastic differentiation and mineralization is unknown, we investigated the role of RBP1 in these processes.MethodsTo create a cell model with suppressed RBP1 expression, primary calvarial osteoblasts were infected with a shRNA lentiviral vector specific for mouse RBP1 (CalOB-ΔRBP1) or a scrambled control shRNA lentivirus (CalOB-Control). Stable cell lines were generated and their mineralization potential was determined using osteoblastic differentiation medium, Alizarin Red staining, and quantitative PCR (QPCR) analyses. Runx2 coactivation by RBP1 was determined through the use of transient transfection assays.ResultsStable expression of the RBP1 shRNA lentivirus in CalOB-ΔRBP1 cells resulted in a 65-70% suppression of RBP1 expression. Osteoblastic mineralization assays demonstrated that suppression of RBP1 results in a potent delay in osteoblastic nodule formation in the CalOB-ΔRBP1 cells with a concomitant decrease in the expression of the osteogenic transcription factors Runx2 and osterix, along with decreases in BMP2, alkaline phosphatase, osteocalcin and bone sialoprotein. Regulation of Runx2 expression by RBP1 was shown to be mediated through the proximal P2 Runx2 promoter. Furthermore, RBP1 was demonstrated to be a potent coactivator of Runx2 transcriptional activity on two known Runx2 reporter constructs. These data suggest that the expression and activity of Runx2 is critically dependent on the presence of RBP1.ConclusionsThis study is the first to demonstrate that RBP1 is an important mediator of the osteoblastic phenotype and clearly defines RBP1 as a novel transcription factor involved in the well known Runx2-osterix transcriptional cascade. As such, the effects of RBP1 on these processes are mediated through both regulation of Runx2 expression and transcriptional activity.
Highlights
Numerous transcription factors are involved in the establishment and maintenance of the osteoblastic phenotype, such as Runx2, osterix and Dlx5
Generation of a mouse calvarial osteoblast cell model with suppressed retinoblastoma binding protein-1 (RBP1) expression Numerous transcription factors are involved in the establishment and maintenance of osteoblastic differentiation such as Runx2, osterix and Dlx5 [16]
A proliferation assay was performed on the Calvarial Osteoblasts (CalOB)-Control and CalOB-ΔRBP1 cell lines since RBP1 has been implicated in cell cycle regulation [5,6], no difference in proliferative capacity of these cells was observed (Figure 1C)
Summary
Numerous transcription factors are involved in the establishment and maintenance of the osteoblastic phenotype, such as Runx, osterix and Dlx. The transcription factor retinoblastoma binding protein-1 (RBP1) was recently identified as an estrogen regulated gene in an osteosarcoma cell model. Retinoblastoma binding protein-1 (RBP1) is a member of the AT-rich interaction domain (ARID) superfamily, known as ARID4A, that is involved in the repression of E2F-dependent transcription and cellular proliferation [5,6] through direct binding with the pocket domain of retinoblastoma (pRb) [7]. This repressive activity is largely due to a RBP1-mediated recruitment of mSin3A/. Our laboratory has recently identified RBP1 as an estrogen-regulated gene in a U2OS osteosarcoma cell model expressing the estrogen receptors (ER) ERα and ERβ [11,12,13]
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