Dysregulated BMP Signaling and Enhanced Osteogenic Differentiation of Connective Tissue Progenitor Cells From Patients With Fibrodysplasia Ossificans Progressiva (FOP)

Abstract

The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells.Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder. Materials and Methods:We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells. Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen-activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand-independent BMP signaling and ligand-dependent hyper-responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells. Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand-stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification.

Insights from a rare genetic disorder of extra-skeletal bone formation, fibrodysplasia ossificans progressiva (FOP)

“…Consistent with constitutive signaling in FOP cells that was suggested by the increased levels of basal p38MAPK activation, we found that expression levels of known BMP transcriptional targets are also increased [51,53]. Analyses using SHED cells were consistent with these LCL studies, and also demonstrated ligand-independent BMP signaling and liganddependent hyper-responsiveness to BMP in cells from FOP patients [45]. SHED cells, which are amenable to osteogenic differentiation assays, additionally showed that FOP SHED cells expressed higher levels of alkaline phosphatase (ALP) and Runx2 mRNAs and more rapidly differentiated to an osteogenic phenotype.…”

“…These highly proliferative and adherent cells are an important cell system to investigate changes in cell differentiation potential of FOP cells [45,46].…”

TGF-β Family Signaling in Connective Tissue and Skeletal Diseases

“…At birth, only minor skeletal abnormalities occur in FOP patients, with congenital big toe malformation as the most characteristic feature. Most cases of FOP are caused by a c.617G.A point mutation in the ACVR1 gene, which encodes the ACVR1/ALK-2 type I receptor and confers a moderate gain-of-function in this BMP type I receptor (Shafritz et al 1996;de la Pena et al 2005;Fiori et al 2006;Shore et al 2006;Billings et al 2008;Fukuda et al 2009;Kaplan et al 2009;Shen et al 2009;Chaikuad et al 2012;Culbert et al 2014). Signaling through the ALK-2 receptor occurs in chondrocytes and osteoblasts, and is required for early stages of chondrogenesis (Culbert et al 2014).…”

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.…”