Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-ß1 (TGF-ß1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

Smad4-dependent transforming growth factor-ß family signaling regulates the differentiation of dental epithelial cells in adult mouse incisors.

Teeth consist of two major tissues, enamel and dentin, which are formed during development by epithelial and mesenchymal cells, respectively. Rodent incisors are useful experimental models for studying the molecular mechanisms of tooth formation because they are simultaneously growing in not only embryos but also adults. Members of the transforming growth factor-ß (TGF-ß) family regulate epithelial-mesenchymal interactions through an essential coactivator, Smad4. In the present study, we established Smad4 conditional knockout (cKO) mice and examined phenotypes in adult incisors. Smad4 cKO mice died with severe anemia within one month. Phosphorylated Smad1/5/9 and Smad2/3 were detected in epithelial cells in both control and Smad4 cKO mice. Disorganized and hypoplastic epithelial cells, such as ameloblasts, were observed in Smad4 cKO mice. Moreover, alkaline phosphatase expression and iron accumulation were reduced in dental epithelial cells in Smad4 cKO mice. These findings suggest that TGF-ß family signaling through Smad4 is required for the differentiation and functions of dental epithelial cells in adult mouse incisors.

Effects of FKBP12 and type II BMP receptors on signal transduction by ALK2 activating mutations associated with genetic disorders.

Various substitution mutations in ALK2, a transmembrane serine/threonine kinase receptor for bone morphogenetic proteins (BMPs), have been identified in patients with genetic disorders such as fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine glioma (DIPG) and heart defects. In this study, we characterized the ALK2 mutants R258G, G328V and F246Y, which were identified in patients with severe FOP, DIPG and unusual hereditary skeletal dysplasia, respectively. Both R258G and G328V were gain-of-function mutations, but F246Y was equivalent to wild-type ALK2. We also examined the effect of the suppressor FKBP12 on the signal transduction of a further 14 ALK2 mutations associated with FOP and/or DIPG. To varying extents FKBP12 over-expression suppressed the basal signaling induced by thirteen of the ALK2 mutants, whereas PF197-8L was uniquely resistant. In the PF197-8L mutant, the modelled ALK2 residue L197 induced a steric clash with the D36 residue in FKBP12 and dissociated their interaction. The co-expression of BMP type II receptors or stimulation with ligands relieved the suppression by FKBP12 by disrupting the interaction between mutant ALK2 and FKBP12. Taken together, FKBP12 binds to and suppresses mutant ALK2 proteins associated with FOP and DIPG, except for PF197-8L.