Retinoblastoma binding protein-1 (RBP1) is a Runx2 coactivator and promotes osteoblastic differentiation.
BACKGROUND
Numerous 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.


METHODS
To create a cell model with suppressed RBP1 expression, primary calvarial osteoblasts were infected with a shRNA lentiviral vector specific for mouse RBP1 (CalOB-DeltaRBP1) 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.


RESULTS
Stable expression of the RBP1 shRNA lentivirus in CalOB-DeltaRBP1 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-DeltaRBP1 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.


CONCLUSIONS
This 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.
Monroe
David G
DG
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. Monroe.David@mayo.edu
Hawse
John R
JR
Subramaniam
Malayannan
M
Spelsberg
Thomas C
TC
D000818:Animals; D002113:Calcification, Physiologic / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology*; D054840:Retinol-Binding Proteins, Cellular / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
D050659:Core Binding Factor Alpha 1 Subunit; C518457:Rbp1 protein, mouse; D054840:Retinol-Binding Proteins, Cellular; C493716:Runx2 protein, mouse; D014157:Transcription Factors