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Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts.

ABSTRACT: CCAAT/enhancer-binding proteins (C/EBPs) as well as bone morphogenic proteins (BMPs) play essential roles in mammalian cell differentiation in shaping adipogenic and osteoblastic lineages in particular. Recent evidence suggested that adipocytes and osteoblasts share a common mesenchymal precursor cell phenotype. Yet, the molecular details underlying the decision of adipocyte versus osteoblast differentiation as well as the involvement of C/EBPs and BMPs remains elusive. We have engineered C2C12 cells for dual-regulated expression of human C/EBP-alpha and BMP-2 to enable independent transcription control of both differentiation factors using clinically licensed antibiotics of the streptogramin (pristinamycin) and tetracycline (tetracycline) classes. Differential as well as coordinated expression of C/EBP-alpha and BMP-2 revealed that (i) C/EBP-alpha may differentiate C2C12 myoblasts into adipocytes as well as osteoblasts, (ii) BMP-2 prevents myotube differentiation, (iii) is incompetent in differentiating C2C12 into osteoblasts and (iv) even decreases C/EBP-alpha's osteoblast-specific differentiation potential but (v) cooperates with C/EBP-alpha on adipocyte differentiation, (vi) osteoblast formation occurs at low C/EBP-alpha levels while adipocyte-specific differentiation requires maximum C/EBP-alpha expression and that (vii) BMP-2 may bias the C/EBP-alpha-mediated adipocyte versus osteoblast differentiation switch towards fat cell formation. Dual-regulated expression technology enabled precise insight into combinatorial effects of two key differentiation factors involved in adipocyte/osteoblast lineage control which could be implemented in rational reprogramming of multipotent cells into desired cell phenotypes tailored for gene therapy and tissue engineering.

SUBMITTER: Fux C

PROVIDER: S-EPMC373304 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts.

Fux Cornelia C Mitta Barbara B Kramer Beat P BP Fussenegger Martin M

Nucleic acids research 20040102 1

CCAAT/enhancer-binding proteins (C/EBPs) as well as bone morphogenic proteins (BMPs) play essential roles in mammalian cell differentiation in shaping adipogenic and osteoblastic lineages in particular. Recent evidence suggested that adipocytes and osteoblasts share a common mesenchymal precursor cell phenotype. Yet, the molecular details underlying the decision of adipocyte versus osteoblast differentiation as well as the involvement of C/EBPs and BMPs remains elusive. We have engineered C2C12 ...[more]

PMID: 14704358

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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

Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts.

Publications

Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts.

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