BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype.
RUNX2 expression in mesenchymal cells induces osteoblast differentiation and bone formation. BMP blocking agents were used to show that RUNX2-dependent osteoblast differentiation and transactivation activity both require BMP signaling and, further, that RUNX2 enhances the responsiveness of cells to BMPs.


BACKGROUND
BMPs and the RUNX2 transcription factor are both able to stimulate osteoblast differentiation and bone formation. BMPs function by activating SMAD proteins and other signal transduction pathways to stimulate expression of many target genes including RUNX2. In contrast, RUNX2 induces osteoblast-specific gene expression by directly binding to enhancer regions in target genes. In this study, we examine the interdependence of these two factors in controlling osteoblast differentiation in mesenchymal progenitor cells.


METHODS
C3H10T1/2 mesenchymal cells and primary cultures of marrow stromal cells were transduced with a RUNX2 adenovirus and treated with BMP blocking antibodies or the natural antagonist, NOGGIN. Osteoblast differentiation was determined by assaying alkaline phosphatase and measuring osteoblast-related mRNA using quantitative RT/PCR. Activation of BMP-responsive signal transduction pathways (SMAD, extracellular signal-regulated kinase [ERK], p38, and c-jun-N-terminal kinase [JNK]) was assessed on Western blots.


CONCLUSIONS
C3H10T1/2 cells constitutively synthesize BMP2 and 4 mRNA and protein, and this BMP activity is sufficient to activate basal levels of SMAD phosphorylation. Inhibition of BMP signaling was shown to disrupt the ability of RUNX2 to stimulate osteoblast differentiation and transactivate an osteocalcin gene promoter-luciferase reporter in C3H10T1/2 cells. BMP blocking antibodies also inhibited RUNX2-dependent osteoblast differentiation in primary cultures of murine marrow stromal cells. Conversely, RUNX2 expression synergistically stimulated BMP2 signaling in C3H10T1/2 cells. However, RUNX2 did not increase the ability of this BMP to activate SMAD, ERK, p38, and JNK pathways. This study shows that autocrine BMP production is necessary for the RUNX2 transcription factor to be active and that BMPs and RUNX2 cooperatively interact to stimulate osteoblast gene expression.
Phimphilai
Mattabhorn
M
Department of Biological Chemistry, University of Michigan School of Medicine, Ann Arbor, Michigan 48109-1078, USA.
Zhao
Zhouran
Z
Boules
Heidi
H
Roca
Hernan
H
Franceschi
Renny T
RT
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; C493709:RUNX2 protein, human; D011994:Recombinant Proteins; D051785:Smad Proteins; D016212:Transforming Growth Factor beta