Abstract
Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin‐A. Binding of Activin‐A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin‐A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis‐inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin‐A on the PLF. PLF‐induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14‐positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony‐stimulating factor (M‐CSF) and receptor activator of nuclear factor kappa‐B ligand (RANK‐L). Although Activin‐A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID‐1) expression only in FOP PLF. Activin‐A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M‐CSF and dendritic cell‐specific transmembrane protein (DC‐STAMP) expression. Activin‐A also inhibited osteoclast formation in M‐CSF and RANK‐L mediated monocultures of CD14+ cells by inhibiting their proliferation.This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin‐A‐mediated HO, this study shows that Activin‐A may also inhibit osteoclast formation, thereby further promoting HO formation.
Highlights
Fibrodysplasia ossificans progressiva (FOP) is a severe genetic disease, characterized by progressive heterotopic bone formation by which muscles, tendons, and ligaments are converted into bone (Bravenboer et al, 2015; Kaplan et al, 2008; Pignolo, Shore, & Kaplan, 2011)
We have recently reported the use of periodontal ligament fibroblasts (PLF) as a tool to study osteoclastogenesis in FOP, where a stronger inhibitory effect of TGF‐β inhibitor on osteoclast formation was observed in FOP fibroblasts‐mediated osteoclast formation
For the RNA isolation cells were cultured under different conditions; PLF were cultured in a 48 wells plate, 1.5 × 104 cells/well, samples were taken after 24 hr incubation without or with 50 ng/ml Activin‐A
Summary
Fibrodysplasia ossificans progressiva (FOP) is a severe genetic disease, characterized by progressive heterotopic bone formation by which muscles, tendons, and ligaments are converted into bone (Bravenboer et al, 2015; Kaplan et al, 2008; Pignolo, Shore, & Kaplan, 2011). FOP is caused by mutations in the gene encoding the Activin receptor type 1 (ACVR1)/activin kinase 2 bone morphogenetic protein (BMP) type 1 receptor, most frequently by the single nucleotide 617G > A mutation This results in the replacement of the amino acid arginine by histidine (R206H; Pignolo et al, 2011; Shore et al, 2006). It has been shown that the mutation results in an increased responsiveness of the receptor to Activin‐A (Hatsell et al, 2015; Hino et al, 2015) This TGF‐β superfamily ligand is known to induce SMAD2/3 signaling (Pearsall et al, 2008), and normally inhibits BMP signaling through ACVR1 (Olsen et al, 2015). We hypothesize that Activin‐A reduces the osteoclastogenesis‐ inducing capacity of the PLF that express the mutant R206H ACVR1 resulting in less bone resorption
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