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Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling.

ABSTRACT: Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of "soft" tissue from which cells can easily be isolated and studied, the "hard" mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors); and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP) receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

SUBMITTER: Zouani OF

PROVIDER: S-EPMC3773333 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling.

Zouani Omar F OF Rami Lila L Lei Yifeng Y Durrieu Marie-Christine MC

Biology open 20130703 9

Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of "soft" tissue from which cells can easily be isolated and studied, the "hard" mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation u ...[more]

PMID: 24143273

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Project description:BackgroundBone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway.Methodology/principal findingsHere we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression.Conclusions/significanceOur study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis.

Dataset Information

Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling.

Publications

Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling.

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