Molecular Consequences of the ACVR1R206H Mutation of Fibrodysplasia Ossificans Progressiva

Abstract

Fibrodysplasia ossificans progressiva (FOP), a rare genetic and catastrophic disorder characterized by progressive heterotopic ossification, is caused by a point mutation, c.617G>A; p.R206H, in the activin A receptor type 1 (ACVR1) gene, one of the bone morphogenetic protein type I receptors (BMPR-Is). Although altered BMP signaling has been suggested to explain the pathogenesis, the molecular consequences of this mutation are still elusive. Here we studied the impact of ACVR1 R206H mutation on BMP signaling and its downstream signaling cascades in murine myogenic C2C12 cells and HEK 293 cells. We found that ACVR1 was the most abundant of the BMPR-Is expressed in mesenchymal cells but its contribution to osteogenic BMP signal transduction was minor. The R206H mutant caused weak activation of the BMP signaling pathway, unlike the Q207D mutant, a strong and constitutively active form. The R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-␤ or BMP signaling. The decreased binding affinity of FKBP1A to the mutant R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state R206H ACVR1 protein levels. Interestingly, while WT ACVR1 and FKBP1A were broadly distributed in plasma membrane and cytoplasm without BMP-2 stimulation and then localized in plasma membrane on BMP-2 stimulation, R206H ACVR1 and FKBP1A were mainly distributed in plasma membrane regardless of BMP-2 stimulation. The impaired binding to FKBP1A and an altered subcellular distribution by R206H ACVR1 mutation may result in mild activation of osteogenic BMP-signaling in extraskeletal sites such as muscle, which eventually lead to delayed and progressive ectopic bone formation in FOP patients.

Role of matrix metalloproteinase-10 in the BMP-2 inducing osteoblastic differentiation

“…The mechanisms by which ALK2(R206H) transfection induces MMP-10 levels in C2C12 cells are still unknown in the present study. BMP family proteins, including BMP-2, 4, 6 and 7, affect cells through ALK2, and the ALK2 (R206H) mutation mildly activates BMP signaling, as previously reported [4]. Although the most crucial ligand of ALK2 for the pathogenesis of FOP have not been determined yet, our preliminary study revealed that exogenous BMP-2 addition did not induce the levels of MMP-10 mRNA in C2C12 cells (data not shown).…”

“…The previous studies showed that ALK2 (R206H) constitutively activates BMP signaling through Smad1/5/8, thereby contributing to the ectopic bone formation in FOP, a disease characterized by marked heterotopic ossification in muscle tissues [1][2][3][4]. We therefore performed a comparative DNA microarray analysis between empty vector-and ALK2 (R206H)-stably transfected mouse myoblastic C2C12 cells to anism of MMP-10 effects on the differentiation of myoblastic cells into osteoblasts.…”

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

“…ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11,12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

“…To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12)(13)(14)(15)(16)(17)(18)(19)(20), mouse embryonic fibroblasts derived from Alk2 R206H/+ mice (21,22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24,25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2 R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter.…”

Genetic abnormalities in Fibrodysplasia Ossificans Progressiva

“…They suggested that miR-148a is an important mediator of ACVR1, thus offering a new potential target for the development of therapeutic agents against FOP (Song et al, 2012). According to Song et al (2010), the p.R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-beta or BMP signaling. The decreased binding affinity of FKBP1A to the mutant p.R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state p.R206H ACVR1 protein levels.…”