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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

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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 3

Smad8 is activated by TGF-β/BMP type I receptors.

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Smad8 exhibits a low activation potential by various ligands of the TGFβ...
(A) Schematic representation of WT Smads and the Smad domains used for functional studies in HEK 293T and C3H10T1/2 cells. MH1 and MH2 are the major conserved Smad domains; “L” shows the linker region between them. The linker region is considerably smaller in Smad8 than in Smad1. (B) Results of reporter assays in HEK 293T cells, in which a GAL4 reporter with the GAL4 DNA-binding domain (GAL4DBD) fused to various forms of Smad proteins was used. Results are expressed as relative luciferase units normalized to β-gal activity and presented as percent of Smad L+MH2, which was arbitrarily set as 100%. Pooled data from at least 3 independent experiments are presented. Smad8 MH2 and L+MH2 domains exhibited a constitutive active transactivating potential comparable to that of Smad1. (C) Activation potential of Smad8 and Smad1 signalling mediators by constitutively active TGF-β/BMP receptors (ALK1–ALK7) in HEK 293T cells. FLAG-tagged Smad8 or Smad1 was transiently coexpressed with constitutively active HA-tagged type I receptors. Expression of all type I receptors was mediated by the identical vector (pcDNA3). Expression rate of the receptors was determined by Western blotting using anti-HA antibodies. Receptor bands indicate variations in the glycosylation of the ectodomain. Type I receptor–dependent phosphorylation of Smads was shown by anti-pSmad1, -5, and -8 antibodies, which also react with phosphorylated Smad8. Smad1 and Smad8 were phosphorylated by most type I receptors. In contrast to Smad1, Smad8 was also efficiently phosphorylated by ALK4 and ALK7 (TGFβ1 receptors).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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