Gsα, the alpha stimulatory subunit of heterotrimeric G proteins that activates downstream signaling through the adenylyl cyclase and cAMP/PKA pathway, plays an important role in bone development and remodeling. The role of Gsα in mesenchymal stem cell (MSC) differentiation to osteoblasts has been demonstrated in several mouse models of Gsα inactivation. Previously, using mice with heterozygous germline deletion of Gsα (Gnas), we identified a novel additional role for Gsα in bone remodeling, and showed the importance of Gnas in maintaining bone quality by regulating osteoclast differentiation and function. In this study, we show that postnatal deletion of Gsα (CreERT2;Gnas) leads to reduction in trabecular bone quality parameters and increased trabecular osteoclast numbers. Furthermore, mice with deletion of Gsα specifically in cells of the macrophage/osteoclast lineage (LysM-Cre;Gnas) showed reduced trabecular bone quality and increased trabecular osteoclasts, but to a reduced extent compared to the CreERT2;Gnas global knockout. This demonstrates that while Gsα has a cell autonomous role in osteclasts in regulating bone quality, Gsα expression in other cell types additionally contribute. In both of these mouse models, cortical bone was more subtly affected than trabecular bone. Our results support that Gsα is required postnatally to maintain trabecular bone quality and that Gsα function to maintain trabecular bone is regulated in part through a specific activity in osteoclasts.

Introduction. Nonhereditary heterotopic ossification (NHO) is a common complication of trauma. Progressive osseous heteroplasia (POH) and fibrodysplasia ossificans progressiva (FOP) are rare genetic causes of heterotopic bone. In this article, we detail the vascular patterning associated with genetic versus NHO. Methods. Vascular histomorphometric analysis was performed on patient samples from POH, FOP, and NHO. Endpoints for analysis included blood vessel (BV) number, area, density, size, and wall thickness. Results. Results demonstrated conserved temporal dynamic changes in vascularity across all heterotopic ossification lesions. Immature areas had the highest BV number, while the more mature foci had the highest BV area. Most vascular parameters were significantly increased in genetic as compared with NHO. Discussion. In sum, both genetic and NHO show temporospatial variation in vascularity. These findings suggest that angiogenic pathways are potential therapeutic targets in both genetic and nonhereditary forms of heterotopic ossification.

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Bone morphogenetic protein (BMP) signaling is critical in skeletal development. Overactivation can trigger heterotopic ossification (HO) as in fibrodysplasia ossificans progressiva (FOP), a rare, progressive disease of massive HO formation. A small subset of FOP patients harboring the causative ACVR1 R206H mutation show strikingly mild or delayed-onset HO, suggesting that genetic variants in the BMP pathway could act as disease modifiers. Whole-exome sequencing of one such patient identified BMPR1A R443C and ACV-R2A V173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1 R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1 R206H signaling. Overexpression of BMPR1A R443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2A V173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1 R206H HEK cells decreased ligand-stimulated BMP signaling but did not affect dorsoventral patterning in Tg(ACVR1-R206Ha) zebrafish. ACVR2A knockdown decreased only AA-stimulated signaling in ACVR1 R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1 R206H HEK cells decreased basal and ligand-stimulated signaling, and co-knockdown/knockout (bmpr1aa/ab; acvr2aa/ab) decreased Tg (ACVR1-R206Ha) zebrafish ventralization phenotypes. Our functional studies showed that knockdown of wild-type BMPR1A andThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Fibrodysplasia ossificans progressiva (FOP, MIM# 135100) is an autosomal‐dominant genetic disorder, caused by heterozygous mutations in the ACVR1 gene. While most with FOP have the same single‐nucleotide substitution (c.617G>A; R206H), occasional variant mutations in ACVR1 have also been identified. The defining clinical features of FOP are malformations of the great toes and progressive heterotopic (extraskeletal) ossification (HO). However, the clinical presentations among FOP patients vary and, at least in some cases, there appear to be genotype–phenotype correlations with specific ACVR1 mutations, even among the small number of patients. FOP‐associated ACVR1 mutations are activating mutations that enhance signalling through the BMP‐pSmad1/5 signalling pathway and direct the induction of cartilage and bone cell differentiation to form ectopic bone in postnatal soft connective tissues. Recent studies examining the molecular mechanisms, reveal that the mutant ACVR1 receptors have lost the normal constraints that regulate the activation of the ACVR1 receptor signalling complex. Key Concepts Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder of heterotopic ossification. FOP is caused by heterozygous activating mutations in the ACVR1 gene. Most FOP patients share the identical ACVR1 c.617G>A mutation that causes a single amino acid substitution (R206H). Additional variant mutations have been identified in ACVR1 and are associated with distinct clinical manifestations in some cases. ACVR1 is a type I receptor in the BMP signalling pathway, an important pathway in regulating bone formation. FOP mutant ACVR1 receptors have lost regulatory constraints that control how downstream signalling is activated.

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Heterotopic ossification (HO), the formation of bone outside of the skeleton, occurs in response to severe trauma and in rare genetic diseases such as progressive osseous heteroplasia (POH). In POH, which is caused by inactivation of GNAS, a gene that encodes the alpha stimulatory subunit of G proteins (Gsα), HO typically initiates within subcutaneous soft tissues before progressing to deeper connective tissues. To mimic POH, we used conditional Gnas-null mice which form HO in subcutaneous tissues upon Gnas inactivation. In response to Gnas inactivation, we determined that prior to detection of heterotopic bone, dermal adipose tissue changed dramatically, with progressively decreased adipose tissue volume and increased density of extracellular matrix over time. Upon depletion of the adipose tissue, heterotopic bone progressively formed in those locations. To investigate the potential relevance of the tissue microenvironment for HO formation, we implanted Gnas-null or control mesenchymal progenitor cells into Gnas-null or control host subcutaneous tissues. We found that mutant cells in a Gnas-null tissue environment induced a robust HO response while little/no HO was detected in control hosts. Additionally, a Gnas-null tissue environment appeared to support the recruitment of control cells to heterotopic bone, although control cell implants were associated with less HO formation compared to mutant cells. Our data support that Gnas inactivation alters the tissue microenvironment to influence mutant and wild-type progenitor cells to contribute to HO formation.