Activin-A Induces Fewer, but Larger Osteoclasts From Monocytes in Both Healthy Controls and Fibrodysplasia Ossificans Progressiva Patients

Abstract

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease characterized by heterotopic ossification (HO) that occurs in muscle tissue, tendons, and ligaments. The disease is caused by mutations in the Activin receptor type I (ACVR1) gene resulting in enhanced responsiveness to Activin-A. Binding of this molecule to the mutated receptor induces HO. Bone metabolism normally requires the coupled action of osteoblasts and osteoclasts, which seems to be disturbed during HO. We hypothesize that Activin-A may also counteract the formation of osteoclasts in FOP patients. In this study we investigated the effect of Activin-A on osteoclast differentiation of CD14+ monocytes from FOP patients and healthy controls. The lymphocytic and monocytic cell populations were determined by FACS analysis. Expression of the mutated R206H receptor was assessed and confirmed by allele specific PCR. The effect of Activin-A on osteoclastogenesis was assessed by counting the number and size of multinucleated cells. Osteoclast activity was determined by culturing the cells on Osteo Assay plates. The influence of Activin-A on expression of various osteoclast related genes was studied with QPCR. Blood from FOP patients contained similar percentages of classical, intermediate, or non-classical monocytes as healthy controls. Addition of Activin-A to the osteoclastogenesis cultures resulted in fewer osteoclasts in both control and FOP cultures. The osteoclasts formed in the presence of Activin-A were, however, much larger and more active compared to the cultures without Activin-A. This effect was tempered when the Activin-A inhibitor follistatin was added to the Activin-A containing cultures. Expression of osteoclast specific genes Cathepsin K and TRAcP was upregulated, gene expression of osteoclastogenesis related genes M-CSF and DC-STAMP was downregulated by Activin-A. Since Activin-A is a promising target for inhibiting the formation of HO in FOP, it is important to know its effects on both osteoblasts and osteoclasts. Our study shows that Activin-A induces fewer, but larger and more active osteoclasts independent of the presence of the mutated ACVR1 receptor. When considering FOP as an Activin-A driven disease that acts locally, Schoenmaker et al. Activin-A and Osteoclasts in FOP our findings suggest that Activin-A could cause a more pronounced local resorption by larger osteoclasts. Thus, when targeting Activin-A in patients with neutralizing antibodies, HO formation could potentially be inhibited, and osteoclastic activity could be slightly reduced, but then performed dispersedly by more and smaller osteoclasts.

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“…Some of them were related to osteoclast differentiation, such as activin receptor type 2A (ACVR2A), activin receptor type 1c (ACVR1C, also known as ALK7), B‐cell translocation gene 2 (BTG2, also known as TIS21), and interferon‐related developmental regulator 1 (IFRD1). Activin‐A binds to activin receptor type II (ACVR2) and activin receptor type I (ACVR1) sequentially and activated ACVR1 phosphorylates SMAD2/3, leading to the expression of target genes and osteoclast differentiation (Kajita et al., 2018 ; Morianos et al., 2019 ; Schoenmaker et al., 2020 ). Also, the expression levels of BTG2 and IFRD1 were increased in mature osteoclasts, and inhibiting BTG2 and IFRD1 reduced the osteoclast differentiation (Iezaki et al., 2016 ; Lee et al., 2002 ).…”

Transcriptomic Differences Underlying the Activin-A Induced Large Osteoclast Formation in Both Healthy Control and Fibrodysplasia Ossificans Progressiva Osteoclasts

“…Apparent cell-type-specific differences in the concentrations and combinations of Type I and Type II bone morphogenic protein receptors between periodontal ligament fibroblasts, and osteoclasts and their precursors could explain such a different response for Activin-A between these cell types [ 28 , 29 , 30 ]. This lack of a disease-specific effect of Activin-A on osteoclasts could suggest that other Activin-A receptors on the osteoclast precursors overruled the mode of action of Activin-A in binding to ACVR1, albeit that ACVR1 is transcribed in osteoclasts and that the mutated form was transcribed at a ninefold higher level compared with the non-mutated form in osteoclasts derived from FOP patients [ 14 ]. ACVR1B, ACVR2A and ACVR2B were not regulated by Activin-A, whereas ACVR1C was downregulated.…”

“…Among the general osteoclast biology terms, our results identified Activin-A upregulated genes that could be associated with enhanced fusion during the formation of osteoclasts. Together with the downregulated genes that are associated with the negative regulation of cell adhesion and migration, this may explain the formation of the larger and more actively resorbing osteoclasts in the presence of Activin-A [ 14 ]. One of the potential modalities to reduce the formation of HO in FOP is to neutralize Activin-A with specific antibodies.…”

“…The RNA samples used for the RNA sequencing and subsequent qPCR analyses were obtained from the osteoclastogenesis experiment described previously [ 14 ]. Briefly, blood was drawn from six sex- and age-matched controls and patients (2 males, 4 females; age range: 20–68 years; maximal age difference between the controls and FOP patients: 4 years; Table 1 ).…”

“…The role of the ACVR1 R206H mutation on the formation of osteoclasts is unclear, as is the effect of Activin-A on the formation of human osteoclasts in general. In a first report on the role of Activin-A in the formation of osteoclasts, we recently described that Activin-A induces fewer, but larger and more active, osteoclasts regardless of the presence of the ACVR1 R206H mutation in the osteoclasts’ precursors [ 14 ]. We described that the osteoclasts that were formed in the presence of Activin-A were at least fourfold larger.…”