BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38.
ABSTRACT Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation.
Article: Osteoblast proliferation and differentiation on a barrier membrane in combination with BMP2 and TGFβ1.[show abstract] [hide abstract]
ABSTRACT: OBJECTIVES: Bioresorbable collagen membranes are routinely utilized in guided bone regeneration to selectively direct the growth and repopulation of bone cells in areas of insufficient volume. However, the exact nature by which alveolar osteoblasts react to barrier membranes as well as the effects following the addition of growth factors to the membranes are still poorly understood. The objective of the present study was therefore to investigate the effect of a bioresorbable collagen membrane soak-loaded in growth factors bone morphogenetic protein 2 (BMP2) or transforming growth factor β1 (TGFβ1) on osteoblast adhesion, proliferation, and differentiation. MATERIAL AND METHODS: Prior to experimental seeding, membranes were soaked in either BMP2 or TGFβ1 at a concentration of 10 ng/ml for 5 min. RESULTS: Human osteoblasts adhered to all soak-loaded membranes as assessed by scanning electron microscopy. Growth factors BMP2 and TGFβ1 increased osteoblast proliferation at 3 or 5 days post-seeding when compared to control collagen membranes. Analysis of real-time PCR revealed that administration of BMP2 increased osteoblast differentiation markers such as osterix, collagen I, and osteocalcin. BMP2 also increased mineralization of primary osteoblasts as demonstrated by alizarin red staining when compared to control and TGFβ1 soak-loaded membranes. CONCLUSION: The combination of a collagen barrier membrane with growth factors TGFβ1 and BMP2 significantly influenced adhesion, proliferation, and differentiation of primary human osteoblasts. CLINICAL RELEVANCE: The described in vitro effects following the combination of collagen barrier membranes with growth factors TGFβ1 and BMP2 provide further biologic support for the clinical application of this treatment strategy in guided bone regeneration procedures.Clinical Oral Investigations 06/2012; · 2.36 Impact Factor
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ABSTRACT: Mechanical stimulation can influence differentiation pathways of stem cells, and may therefore provide improved control of lineage specifications for clinical applications. Oscillatory mechanical stimulation at relatively low frequencies (0.01 Hz) has recently been shown to suppress adipogenic differentiation of mesenchymal stem cells, indicating that the range of effective stimulation frequencies is not limited to those associated with locomotion, circulation, and respiration. We hypothesized that relatively low frequency mechanical stimulation (0.01 Hz) can also promote osteogenic cell differentiation of myoblastic C2C12 cells in combination with BMP-2. Results indicate that low frequency mechanical stimulation can significantly enhance osteogenic gene expression, provided that differentiation is initiated by a priming period involving BMP-2 alone. Subsequent application of low frequency mechanical stimulation appears to act synergistically with continued BMP-2 exposure to promote osteogenic differentiation of C2C12 cells, and can even partially compensate for the removal of BMP-2. These effects may be mediated by the ERK and Wnt signalling pathways. Osteogenic induction of C2C12 cells by low frequency mechanical stimulation is therefore critically dependent upon previous exposure to growth factors, and the timing of superimposed BMP-2 and mechanical stimuli can sensitively influence osteogenesis. These insights may provide a technically simple means for control of stem cell differentiation in cell-based therapies, particularly for enhancement of differentiation toward desired lineages.ISRN Stem Cells. 01/2013;
Article: Time series gene expression profiling and temporal regulatory pathway analysis of BMP6 induced osteoblast differentiation and mineralization.[show abstract] [hide abstract]
ABSTRACT: BMP6 mediated osteoblast differentiation plays a key role in skeletal development and bone disease. Unfortunately, the signaling pathways regulated by BMP6 are largely uncharacterized due to both a lack of data and the complexity of the response. To better characterize the signaling pathways responsive to BMP6, we conducted a time series microarray study to track BMP6 induced osteoblast differentiation and mineralization. These temporal data were analyzed using a customized gene set analysis approach to identify temporally coherent sets of genes that act downstream of BMP6. Our analysis identified BMP6 regulation of previously reported pathways, such as the TGF-beta pathway. We also identified previously unknown connections between BMP6 and pathways such as Notch signaling and the MYB and BAF57 regulatory modules. In addition, we identify a super-network of pathways that are sequentially activated following BMP6 induction. In this work, we carried out a microarray-based temporal regulatory pathway analysis of BMP6 induced osteoblast differentiation and mineralization using GAGE method. This novel temporal analysis is more informative and powerful than the classical static pathway analysis in that: (1) it captures the interconnections between signaling pathways or functional modules and demonstrates the even higher level organization of molecular biological systems; (2) it describes the temporal perturbation patterns of each pathway or module and their dynamic roles in osteoblast differentiation. The same set of experimental and computational strategies employed in our work could be useful for studying other complex biological processes.BMC Systems Biology 05/2011; 5:82. · 3.15 Impact Factor