Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells
Andrea Hoffmann, … , Gerhard Gross, Dan Gazit
Andrea Hoffmann, … , Gerhard Gross, Dan Gazit
Published April 3, 2006
Citation Information: J Clin Invest. 2006;116(4):940-952. https://doi.org/10.1172/JCI22689.
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Research Article

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells

Abstract

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Authors

Andrea Hoffmann, Gadi Pelled, Gadi Turgeman, Peter Eberle, Yoram Zilberman, Hadassah Shinar, Keren Keinan-Adamsky, Andreas Winkel, Sandra Shahab, Gil Navon, Gerhard Gross, Dan Gazit

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Figure 4

Smad8 exhibits a low activation potential by various ligands of the TGFβ/BMP family.

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Smad8 is activated by TGF-β/BMP type I receptors. (A) Schematic represen...
(A) FLAG-tagged Smad1 or Smad8 were transiently expressed in C3H10T1/2, and BMP2, TGFβ1, or GDF5 were added for 30 minutes at the indicated concentrations. After Western blotting, ligand-dependent phosphorylation of Smads was shown by antibodies specific for pSmad1, -5, and -8. The expression rate of the Smads was determined by Western blotting using anti-FLAG antibodies. (B) Evaluation of the Smad1 and Smad8 transactivation potential in HEK 293T cells, in which a GAL4 reporter with the GAL4 DNA-binding domain was fused to Smad1WT and Smad8WT proteins as described in Figure 3B. Pooled data from 3 independent experiments are presented. BMP2 led to efficient activation of the GAL4-Smad1 fusion protein, GDF5 was less effective, and TGFβ1 did not exhibit notable activation. The GAL4-Smad8 fusion protein was activated, albeit to a markedly lower extent, by BMP2, but not by TGFβ1 or GDF5. The expression level of the GAL4-Smad fusions was comparable as assessed by Western analyses of cellular extracts and blotting with anti-GAL4 antibodies. (C) Compared with Smad1, Smad8 exhibited a lower BMP2-dependent activation potential in C3H10T1/2. FLAG-tagged Smad1 or Smad8 were transiently expressed in C3H10T1/2, and BMP2 (200 ng/ml) was added for 30 minutes. Top: After Western blotting, BMP2-dependent phosphorylation of Smads is shown by anti-pSmad1, -5, and -8 antibodies. Bottom: Expression rates of the Smads were determined by Western blotting using anti-FLAG antibodies. In the graph at right, the level of BMP2-dependent Smad activation in C3H10T1/2 was evaluated by densitometric scanning.